WO2017110705A1 - Agent for enhancing synapse formation and therapeutic agent for neurodegenerative disease - Google Patents

Abstract

The present invention addresses the problem of developing a drug capable of regrowing synapses, which have been damaged and lost due to a neurodegenerative disease, to thereby provide a medicinal composition for preventing or treating a neurodegenerative disease on the basis of the effect of the aforesaid drug. Provided is a therapeutic agent for a neurodegenerative disease which comprises an agent for enhancing synapse formation, said agent comprising miR132 or miR212, as an active ingredient.

Description

Synapse formation enhancer and neurodegenerative disease therapeutic agent

The present invention relates to a synapse formation enhancer and a therapeutic agent for neurodegenerative diseases including the same as an active ingredient.

A neurodegenerative disease is a progressive neurological disease that develops when a group of neurons in a specific region in the central nervous system or the like gradually disappears. Various neurodegenerative diseases have been reported, but all are intractable diseases and lack effective treatment.

Neurodegenerative diseases are generally likely to occur in the elderly and are considered to be due to aging. In addition, since neurodegenerative diseases have a long incubation period until onset and treatment after onset is generally difficult, preventive measures before onset are regarded as important. In general, the prevention of disease is premised on the elucidation of the pathogenesis of the target disease. However, regarding neurodegenerative diseases, the causative genes have been identified in some inherited familial neurodegenerative diseases, and the onset mechanism is being elucidated at the gene level and protein level. Many of the neurodegenerative diseases described above have not been clarified yet (Non-patent Documents 1 and 2).

In the future aging society, an increase in the number of patients with neurodegenerative diseases such as dementia is expected to become a serious social problem because it will increase medical expenses and increase the burden of nursing care. In addition, the economic and social losses due to the increase in patients with dementia are immeasurable. Therefore, there is an urgent need to develop preventive and therapeutic methods that can prevent the onset of neurodegenerative diseases and, for patients who have already developed, to improve the patient's QOL by suppressing progression and reducing or improving symptoms. It has become.

Currently, in the development of many disease treatments, efforts are being focused on methods for suppressing or inhibiting the activity of pathogenic genes and their gene products that cause their development (Non-Patent Documents 3 to 6). However, the therapeutic drug or treatment method in which the target molecule is specified is limited in the pharmacological effect and therapeutic range to the range in which the molecule is involved. For example, if a drug that targets the aberrant huntingtin protein, which is the causative molecule, is developed as a therapeutic drug for Huntington's disease, Alzheimer It cannot be expected for sick patients. Therefore, it is necessary to elucidate the cause and occurrence mechanism of each neurodegenerative disease and to develop a therapeutic agent or treatment method for the cause. There is no known general-purpose drug that can prevent many neurodegenerative diseases and improve or treat symptoms with one therapeutic drug.

省 Provincial, Neurodegenerative Diseases-Advances in Research and Diagnosis, History of Acupuncture, 2013 Nov. 247 (5) Mitsui J. and Tsuji S., 2014, Biochem Biophys Res Commun. 452 (2): 221-5. Nagai Y., et al., 2007, Brain Nerve. 59 (4): 393-404. Hohjoh H., 2013, Pharmaceuticals (Basel), 6 (4): 522-535. Takahashi M., et al., 2010, Proc Natl Acad Sci USA, 107 (50): 21731-21736 Takahashi M., et al., 2012, Gene Ther. 19 (7): 781-785

The present invention is to develop a drug for re-forming a synapse that has been destroyed and disappeared by a neurodegenerative disease, and to provide a pharmaceutical composition for treating or preventing the neurodegenerative disease by its effect.

In order to solve the above problems, the present inventors searched for factors involved in synapse formation.

¡Synapse formation increases explosively at an early stage after birth. Therefore, it is highly possible that factors involved in synapse formation are rapidly increasing in the early stages of life. Therefore, gene expression profiles of normal mouse brain neurons at various times within the first month of life were created, and genes whose expression increased dramatically and were subsequently maintained were selected as candidate genes. On the other hand, using the model mouse R6 / 2 strain of Huntington's disease, the expression profile at the same time of the candidate gene was verified, and the expression pattern with normal mice was compared. In R6 / 2 mice, synapse formation is likely to be suppressed. As a result, in R6 / 2 mice, miR-132 gene and miR-212 gene were expressed as dramatically as normal mice, and after reaching a plateau, it was significantly decreased without being maintained. (Fig. 1). In an in vitro rescue experiment with miR-132, synapse formation was significantly restored in cells with a pathogenic mutant huntingtin gene. In addition, when miR-132 was introduced into the striatum of R6 / 2 mice, it was revealed that motor function and lifespan improved significantly. Furthermore, it has been found that miR-132 can improve Huntington's disease without directly processing the mutant huntingtin gene or its product. The present invention is based on the new knowledge and provides the following.

(1) A synapse formation enhancer comprising a polynucleotide comprising the base sequences shown in the following (a) to (c) or a transcription product thereof.
(A) the nucleotide sequence represented by SEQ ID NO: 1 or 5,
(B) a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1 or 5, or (c) 95% or more of the base sequence represented by SEQ ID NO: 1 or 5 (2) The synapse formation enhancer according to (1), wherein the polynucleotide is included in a state in which the polynucleotide can be expressed in an expression vector.
(3) A neurodegenerative disease preventive or therapeutic agent comprising the synapse formation enhancer according to (1) or (2) as an active ingredient.
(4) the neurodegenerative disease is Huntington's disease, basal ganglia degeneration, progressive supranuclear palsy, Alzheimer's disease, Lewy body dementia, frontotemporal lobar degeneration, spinocerebellar degeneration, Parkinson's disease, or The therapeutic agent for neurodegenerative disease according to (3), which is amyotrophic lateral sclerosis.

This specification includes the disclosure of Japanese Patent Application No. 2015-248219, which is the basis of the priority of the present application.

The synapse formation enhancer of the present invention can act on nerve cells to re-form synapses that have disappeared due to destruction or dissociation.

The agent for preventing and treating neurodegenerative diseases of the present invention can enhance the synapse formation of nerve cells. The effect can prevent the development of neurodegenerative diseases and improve the cranial nerve function lost due to the neurodegenerative diseases.

Figure 7 shows the expression profiles of miR-132, miR-212 and negative control miR-16 in 5 subregions of the brain of normal mice and neurodegenerative disease model mice. It is a figure which shows the expression level of miR-132 in the striatum of the mouse | mouth processed with viral expression vector AAV9_miR-132 or AAV9_miR-Neg. Data were normalized with the expression level of wild type mice carrying AAV9_miR-Neg as 1. The experiment was performed independently on 6 mice (n = 6). “*” In the figure indicates P <0.05. It is a figure which shows the result of the rotarod test of the wild type mouse | mouth (WT) and R6 / 2 mouse | mouth (HD) which were processed with viral expression vector AAV9_miR-132 or AAV9_miR-Neg. It is a figure which shows the result of the open field test of the mouse | mouth processed with viral expression vector AAV9_miR-132 or AAV9_miR-Neg. “*” In the figure indicates P <0.05. It is a figure which shows the survival curve of the wild type mouse | mouth (WT) and R6 / 2 mouse | mouth (HD) which were processed by viral expression vector AAV9_miR-132 or AAV9_miR-Neg. a is a R6 / 2 mouse introduced with AAV9_miR-Neg, b is an R6 / 2 mouse introduced with AAV9_miR-132, c is a wild type mouse introduced with AAV9_miR-132, d is a wild type mouse introduced with AAV9_miR-Neg Each type mouse is shown. “*” In the figure indicates P <0.05. It is a western blotting showing the amount of synapse formation-related protein (vGluT1, SynI, PSD95) in the mouse brain. A: It is a figure which shows the result of immunocytochemistry. Each stage image shows the same field of view. The arrows in the Merge diagram indicate SynI positive synaptic spines on the neurites. B: A graph showing the number of SynI-positive synaptic spines present on a 10 μm neurite obtained from the result of A. In the figure, “*” indicates P <0.05 and “n.s.” indicates that there is no significant difference “significant difference”. A: It is a figure which shows the immunohistochemistry of the brain in R6 / 2 mouse (HD) which introduce | transduced AAV9_miR-132 or AAV9_miR-Neg. The upper and lower images show the same field of view. In the figure, arrowheads indicate inclusion bodies observed in GFP positive cells. B: A graph showing the number of GFP-positive cells including inclusion bodies in three different visual fields (247 μm × 247 μm / field) per mouse into which AAV9_miR-132 or AAV9_miR-Neg was introduced. In the figure, “n.s.” indicates that there is no significant difference “significant difference”. It is a figure which shows the expression level of the mutant | variant HTT transgene (Transgene) in the R6 / 2 mouse | mouth treated with AAV9_miR-132 or AAV9_miR-Neg. In the upper part of this figure, total mRNA was extracted from the brain of each mouse, RT-PCR was performed using the primers shown in SEQ ID NOs: 11 and 12, and the amplified product was developed on an agarose gel. Ethidium bromide The gel dye | stained with is shown. The lower row shows the expression level of Gapdh (glyceraldehyde-3-phosphate dehydrogenase) gene used as an internal standard. The expression profile of miR-132 in the cerebellum (Crb) of a normal mouse (WT) and a spinocerebellar degeneration model mouse (SCA1) is shown. The expression profile of miR-132 in (A) cerebral cortex (Ctx), (B) midbrain (MB), and (C) striatum of normal mouse (WT) and spinocerebellar degeneration model mouse (SCA1) is shown.

1. 1. Synapse formation enhancer 1-1. Outline | summary The 1st aspect of this invention is a synapse formation enhancer. The synapse formation enhancer of the present invention consists of a specific miRNA or a polynucleotide encoding the same. According to the synapse formation enhancer of the present invention, the formed synapse can be maintained or strengthened, and the destroyed synapse can be reformed.

1-2. Definitions “Synapse” refers to a contact structure for transmission of information between nerve cells or between nerve cells and cells of other species. The “other cells” referred to here mainly correspond to muscle cells (muscle fibers). In the present specification, unless otherwise specified, hereinafter, a synapse refers to a synapse in a narrow sense, that is, a contact structure between nerve cells. Synapses are broadly divided into “chemical synapses” that transmit information between neurons via neurotransmitters and “electrical synapses” that transmit information via ionic current between cells attached by cell adhesion molecules. However, the synapse of this specification may be any synapse. A chemical synapse is preferable.

In this specification, “synapse formation” means that synapses are formed between cells, mainly between nerve cells. Synapse formation between neurons is usually formed between the axon end of a presynaptic cell that transmits an information signal and the dendrite of a post-synaptic cell that receives the signal. By forming higher-order synapses between a plurality of nerve cells, a complex neural network (neural network) found in the brain is formed.

As used herein, “enhancement of synapse formation” means increasing synapse formation, maintaining or strengthening the formed synapse, or reforming a synapse destroyed or dissociated by a disorder such as a neurodegenerative disease And restoring the previous neural network.

1-3. Structure The synapse formation enhancer of this invention is comprised with a polynucleotide. This polynucleotide comprises the miR-132 gene or miR-212 gene, or transcripts thereof.
(1) miR-132 gene The “miR-132 gene” is a gene encoding miR-132 and is composed of DNA in principle. The miR-132 gene has been reported to be involved in neuronal maturation and synaptic stability (Magill, ST, et al., 2010, Proc Natl Acad Sci USA 107, 20382-20387; Wanet, A., et al., 2012, Nucleic Acids Res 40, 4742-4753; Remenyi, J., et al., 2013, PLoS One 8, e62509). In addition, it has been shown that miR-132 gene expression increases dramatically in the early childhood brain and is maintained thereafter (Eda, A., et al., 2011, Gene 485, 46 -52). However, there has been no report on the association with synapse formation and diseases such as neurodegenerative diseases, and the specific function has not been known so far.

The miR-132 gene constituting the synapse formation enhancer of the present invention is a wild-type miR-132 gene encoding wild-type miR-132, or a mutant miR-132 having an activity equal to or higher than that of wild-type miR-132. Includes the encoded mutant miR-132 gene.

Examples of the wild-type miR-132 gene include a human wild-type miR-132 gene consisting of the base sequence represented by SEQ ID NO: 1. Also included are wild-type miR-132 ortholog genes derived from other species having the same activity as the human wild-type miR-132 gene. Specifically, wild-type miR-132 ortholog genes derived from mammals other than humans, and wild-type miR-132 ortholog genes derived from birds, reptiles, or amphibians are applicable. The miR-132 gene is highly conserved among species, and the wild-type miR-132 ortholog gene of many mammals such as mice, Chinese hamsters, and pygmy chimpanzees is the same as the human wild-type miR-132 gene. It is known to have 100% base identity. Moreover, even in reptiles, for example, the wild-type miR-132 gene is 95% or more like the wild-type miR-132 orthologous gene of the green anole (Anolis carolinensis) consisting of the base sequence shown in SEQ ID NO: 2. Has high base identity.

Examples of the mutant miR-132 gene include a mutant gene of the human wild-type miR-132 gene consisting of the base sequence shown in SEQ ID NO: 1. For example, a polynucleotide comprising a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 1, or 95% or more, 97% or more of the nucleotide sequence represented by SEQ ID NO: 1 , A polynucleotide comprising a nucleotide sequence having base identity of 98% or more, or 99% or more, and a nucleic acid fragment comprising a nucleotide sequence complementary to the partial nucleotide sequence of the miR-132 gene comprising the nucleotide sequence represented by SEQ ID NO: 1 And a polynucleotide comprising a base sequence that hybridizes under highly stringent conditions. As used herein, “base identity” refers to aligning the base sequences of two polynucleotides and introducing a gap into one of the base sequences as necessary, so that the base coincidence between both is the highest. The ratio (%) of the same base of the other polynucleotide with respect to the total number of bases of one polynucleotide when it is made higher. % Identity is obtained by using a known program such as homology search program BLAST (Basic local alignment search tool; Altschul, S. F. et al, J. Mol. Biol., 215, 403-410, 1990) Easy to determine. In the present specification, “a plurality of bases” means 2 to 60, 2 to 45, 2 to 30, 2 to 14, 2 to 10, for example, 2 to 8, 2 to 6, Refers to 2-5, 2-4, or 2-3 bases. In the present specification, “high stringent conditions” refers to conditions of high temperature and low salt concentration at which non-specific hybrids are not formed. For example, in the post-hybridization washing, the condition is 1 × SSC or less at 60 ° C. to 68 ° C., preferably 0.1 × SSC or less at 65 ° C. to 70 ° C.

(2) miR-132
“MiR-132” is a transcription product of the miR-132 gene, and is composed of RNA in principle. Specifically, human wild-type miR-132 consisting of the base sequence represented by SEQ ID NO: 3 can be mentioned. Other examples include transcripts of other miR-132 ortholog genes such as the wild-type miR-132 of green anole consisting of the base sequence shown in SEQ ID NO: 4, and transcripts of mutant miR-132 genes.

MiR-132 is a kind of miRNA. miRNA is a single-stranded non-coding RNA having a length of 21 to 23 bases existing in cells and is known to regulate the expression of a target gene by inhibiting the translation of the target gene. miRNA is transcribed from the genome in a precursor (pre-precursor) state called pri-miRNA, then processed into a precursor called pre-miRNA in the nucleus by an endonuclease called Drosha, and then dicer outside the nucleus. It becomes miRNA of mature body by the action of endonuclease called (Bartel DP, 2004, Cell, 116: 281-297). As used herein, miRNA encompasses both miRNA precursors and mature miRNAs. The transcript miR-132 has substantial activity against the target gene.

The target gene of miR-132 is not yet clear. However, in the present specification, miR-132 enhances synapse formation without directly targeting the pathogenicity gene of neurodegenerative disease or its product, and constructs a stronger neural network. It has been shown to enhance the resistance of the product to toxicity.

(3) miR-212 gene “miR-212 gene” is a gene encoding miR-212 and is composed of DNA. The miR-212 gene constitutes the same cluster adjacent to the miR-132 gene on the human genome and is considered to be under the same expression control as the miR-132 gene (Wanet, A., et. al, 2012, Nucleic Acids Res 40, 4742-4753). Similar to the miR-132 gene, expression increases dramatically in the early childhood brain (Eda, A., et al., 2011, Gene 485, 46-52), and the nucleotide sequence between the two genes. Since both have high base identity (82% for human miR-132 gene and miR-212 gene), both genes are considered to be paralogs having the same function caused by gene duplication.

The miR-212 gene constituting the synapse formation enhancer of the present invention is a wild-type miR-212 gene encoding wild-type miR-212 or a mutant miR-212 having an activity equal to or higher than that of wild-type miR-212. It includes the mutant miR-212 gene encoding.

Examples of the wild-type miR-212 gene include a human wild-type miR-212 gene consisting of the base sequence represented by SEQ ID NO: 5. Moreover, the miR-212 ortholog gene derived from other biological species having the same activity as the human wild-type miR-212 gene can also be mentioned. The nucleotide sequence of the miR-212 gene is also highly conserved among species, similar to the miR-132 gene. For example, the wild-type miR-212 ortholog gene of mammals such as mice, rats, oppossums and horses has 100% base identity with the human wild-type miR-212 gene.

Examples of the mutant miR-212 gene include a mutant gene of the human wild-type miR-212 gene consisting of the base sequence shown in SEQ ID NO: 5. For example, a polynucleotide comprising a base sequence in which one or more bases have been deleted, substituted or added in the base sequence represented by SEQ ID NO: 5, 95% or more, 97% or more with respect to the base sequence represented by SEQ ID NO: 5, A polynucleotide comprising a base sequence having base identity of 98% or more, or 99% or more, a nucleic acid fragment comprising a base sequence complementary to a partial base sequence of the miR-212 gene comprising a base sequence represented by SEQ ID NO: 5, and a high A polynucleotide comprising a base sequence that hybridizes under stringent conditions is included.

(4) miR-212
“MiR-212” is a transcription product of the miR-212 gene and is composed of RNA in principle. Specifically, human wild-type miR-212 consisting of the base sequence represented by SEQ ID NO: 6 can be mentioned. In addition, transcripts of other miR-212 ortholog genes and mutant miR-212 genes can be mentioned. miR-212 is also a kind of miRNA, like miR-132.

(5) Expression vector In the synapse formation enhancer of this invention, the said miRNA gene, ie, miR-132 gene and / or miR-212 gene, may be included in the state which can be expressed to an expression vector.

“Expression vector” generally refers to a vector capable of controlling expression of a target gene contained therein. “Expressable state” refers to a state in which a gene of interest can be transcribed in a host cell under predetermined conditions. For example, a state in which a target gene is arranged under the control of a promoter and a terminator is applicable. In this specification, the target gene included in the expression vector is the miR-132 gene and / or the miR-212 gene.

The expression vector can contain control elements necessary for gene expression control as required. Examples of the control element include an enhancer and a poly A addition signal in addition to the above-described essential elements such as a promoter and a terminator.

Promoter must be operable in the host cell. In general, a promoter derived from a host species or a related species is preferred. For example, if the host is human, the promoter is preferably derived from human. As the promoter, an overexpression promoter, a constitutive promoter, a site-specific promoter, a developmental stage-specific promoter, an inducible promoter, or the like is known depending on the expression pattern. Any promoter can be used for the expression vector in the agent for enhancing synapse formation of the present invention. However, if it is a site-specific promoter, a synapse-forming cell, for example, a nerve cell-specific promoter is preferable. In the case of a developmental stage-specific promoter, a promoter corresponding to the developmental stage of the host to be applied is used.

The terminator is not particularly limited as long as it is a sequence capable of terminating the transcription of the gene transcribed by the promoter. Preferred is a terminator derived from the same species as the promoter, and more preferred is a terminator paired with the promoter on the genome of the species from which the promoter is derived.

The expression vector may contain a selection or marker marker gene for confirming that the expression vector has been delivered into the host cell, if necessary. Labeling or selection marker genes include, for example, fluorescent or luminescent reporter genes (eg, luciferase, β-galactosidase, GUS, or GFP), or enzyme genes.

The type of expression vector is not particularly limited as long as it is a plasmid or virus that can be replicated and expressed in a host cell to which the synapse formation enhancer of the present invention is applied. For example, when the host to be applied is human, viruses such as adeno-associated virus (AAV), adenovirus, retrovirus (including lentivirus), Sendai virus and the like can be used. In addition, expression vectors for various hosts commercially available from each life science manufacturer can also be used.

¡Two or more expression cassettes can be contained in a single expression vector in a state where miRNA genes can be expressed.

1-4. Effect The synapse formation-enhancing agent of the present invention has no side effects on nerve cells and can re-form synapses that have disappeared due to destruction or dissociation. Therefore, it can function as a cranial nerve function recovery agent in diseases caused by synaptic destruction or damage.

2. 2. Agents for preventing and treating neurodegenerative diseases 2-1. Outline | summary The 2nd aspect of this invention is a neurodegenerative disease preventive-therapeutic agent. The agent for preventing or treating neurodegenerative diseases of the present invention can prevent or treat a neurodegenerative disease by using the synapse formation enhancing agent of the first aspect as an active ingredient and administering it to a living body.

2-2. Definitions The agent for preventing and treating neurodegenerative diseases of the present invention is a pharmaceutical composition applied for the prevention and treatment of neurodegenerative diseases.

As used herein, “prevention” refers to preventing the onset of a disease (a neurodegenerative disease in this specification). In the present specification, “treatment” refers to alleviating, suppressing, or preventing the progression of symptoms in an onset disease and improving the symptoms.

As used herein, the term “neurodegenerative disease” refers to the formation of abnormal proteinaceous inclusions in specific nerve cells (neurons) or glial cells of the central nervous system, and as a result, nerve cells are gradually invaded. It refers to progressive neurological disease that declines and disappears.

Examples of neurodegenerative diseases that are the subject of the present invention include Huntington's disease (HD; Huntington disease), basal ganglia degeneration (CBD), progressive supranuclear palsy (CBD; PSP; progressive supranuclea palsy), Parkinson's disease (Parkinson's disease), Alzheimer's disease (AD; Alzheimer disease) with cerebral lesions, Lewy body dementia, Frontotemporal lobar degeneration ), Spinocerebellar degeneration with lesions in the cerebellum (SCD; Spinocerebellar Degeneration), and amyotrophic lateral sclerosis (ALS) with lesions in motor neurons.

2-3. Configuration 2-3-1. Active ingredient The neurodegenerative disease preventive and therapeutic agent of the present invention includes the synapse formation enhancer according to the first aspect as an active ingredient. The neurodegenerative disease preventive / therapeutic agent can contain two or more synapse formation enhancers.

The amount (content) of the synaptogenesis enhancer contained in the agent for preventing or treating neurodegenerative diseases is the type of synapse enhancer (DNA or RNA, miR-132 or miR-212, or both), It depends on the type of neurodegenerative disease, the dosage form of the neurodegenerative disease preventive and therapeutic agent, the dose of the neurodegenerative disease preventive and therapeutic agent, and the type of carrier described below. Therefore, it may be determined appropriately in consideration of each condition. Usually, it is adjusted so that an effective amount of a synapse formation enhancer is included in a single dose of a neurodegenerative disease preventive or therapeutic agent. "Effective amount" refers to an amount necessary for the synapse formation enhancer to function as an active ingredient, and an amount that causes little or no harmful side effects on the living body to which the synapse formation enhancer is applied. . This effective amount may vary depending on various conditions such as subject information, route of administration, and number of doses. Ultimately, it is determined by the judgment of a doctor, veterinarian or pharmacist.

Here, “subject” refers to a living body to which a neurodegenerative disease preventive or therapeutic agent is applied. For example, humans, pets (dogs, cats, etc.), livestock (cows, horses, sheep, goats, pigs, chickens, ostriches, etc.), racehorses, laboratory animals (mouse, rats, rabbits, guinea pigs, monkeys, etc.) Applicable. Preferably it is a human. “Subject information” is various individual information of a living body to which a neurodegenerative disease preventive or therapeutic agent is applied. For example, if the subject is a human, it includes age, weight, sex, diet, health status, disease progression and severity, drug sensitivity, presence of concomitant drugs, and the like.

2-3-2. Carrier The neurodegenerative disease prophylactic and therapeutic agent of the present invention can contain a pharmaceutically acceptable carrier as necessary. “Pharmaceutically acceptable carrier” refers to an additive usually used in the field of pharmaceutical technology. Examples thereof include solvents, excipients, binders, disintegrants, fillers, emulsifiers, fluid addition regulators, lubricants, human serum albumin, and the like.

The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of the aqueous solution include physiological saline, isotonic solutions containing glucose and other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.

Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronic, kaolin, silicic acid, or combinations thereof. It is done.

Examples of the binder include starch paste using plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof. Can be mentioned.

Examples of the disintegrant include the starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearin. Examples include acid monoglycerides or salts thereof.

Examples of fillers include petrolatum, the sugar and / or calcium phosphate.

Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

Examples of the flow addition regulator and lubricant include silicate, talc, stearate or polyethylene glycol.

In addition to the above, solubilizers, suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizers that are commonly used in medicine, if necessary , Moisturizers, wetting agents, adsorbents, flavoring agents, disintegration inhibitors, coating agents, colorants, preservatives, preservatives, antioxidants, fragrances, flavoring agents, sweeteners, buffering agents, tonicity agents, etc. Can be included as appropriate.

The above carrier is used for avoiding or suppressing degradation of the synapse formation enhancer, which is an active ingredient, by enzymes in vivo, facilitating formulation and administration methods, and maintaining dosage form and efficacy. Yes, it can be used as needed.

2-3-3. Dosage Form The dosage form of the agent for preventing and treating neurodegenerative diseases of the present invention delivers the synapse formation enhancing agent according to the first aspect, which is an active ingredient, to the target site without being inactivated by degradation or the like, and in vivo The form is not particularly limited as long as the pharmacological effect of the active ingredient can be exhibited.

The disease to which the agent for preventing and treating neurodegenerative diseases is applied is a neurodegenerative disease described later. A site causing a neurodegenerative disease is a site where nerve cells are present, that is, mainly the central nerve, particularly the brain. Therefore, the agent for preventing and treating neurodegenerative diseases can be in any dosage form as long as it can deliver the synapse-enhancing agent of the active ingredient to the target site, more specifically to the nerve cell, with the central nerve, particularly the brain as the target site. Good.

The specific dosage form varies depending on the administration method and / or prescription conditions. Administration methods can be broadly classified into parenteral administration and oral administration, so that dosage forms suitable for each administration method may be used.

If the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically administered via the circulatory system. An example of the liquid agent is an injection. Injections are formulated by mixing in the unit dosage form generally required for pharmaceutical practice, in combination with the excipients, emulsifiers, suspensions, surfactants, stabilizers, pH regulators, etc. as appropriate. Can be

If the administration method is oral administration, preferred dosage forms include solid preparations (including tablets, capsules, drops, lozenges), granules, powders, powders, and liquids (internal solutions, emulsions, syrups). ). If it is a solid preparation, a dosage form with a coating known in the art, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary be able to.

The specific shape and size of each dosage form are not particularly limited as long as the dosage form is within the range of dosage forms known in the art for each dosage form. What is necessary is just to formulate according to the conventional method of the said technical field about the manufacturing method of the neurodegenerative disease prevention-treatment agent of this invention. For example, the method described in Remington's Pharmaceuticals Sciences (Merck Publishing Co., Easton, Pa.) Can be referred to.

2-3-4. Administration Method As described above, the administration method of the agent for preventing and treating neurodegenerative diseases of the present invention can be roughly classified into parenteral administration and oral administration. Oral administration is generally systemic, but parenteral administration can be further subdivided into local and systemic administration. Specifically, for example, local administration by intracerebral injection or systemic administration by circulatory administration such as intravascular injection can be mentioned.

The administration method of the neurodegenerative disease preventive / therapeutic agent of the present invention can be appropriately selected according to the onset or progress of the disease, and may be either local administration or systemic administration. The synapse formation enhancer which is an active ingredient acts on nerve cells. Moreover, as shown in the Example mentioned later, a synapse formation enhancer does not produce a side effect even if it administers to a normal nerve cell. Therefore, it can be locally administered to the brain where the disease occurs because it has no or very little side effects. Local administration is preferably by injection. For systemic administration, for example, intravascular injection or oral administration can be employed. Intravascular injection is convenient in that it can spread the synapse formation enhancer throughout the body through the bloodstream. However, in this case, it is preferable that the synapse formation enhancer comprising the polypeptide as a constituent component is not decomposed in the blood, and passes through the brain barrier to be delivered to the target brain neurons. For example, a synapse-enhancing agent that includes miR-132 gene or miR-212 gene in type 1, type 2, or type 5 adeno-associated virus (AAV) vector that targets neuronal cells, and nucleic acid degradation by AAV particles Can be delivered to brain neurons.

2-4. Applicable disease The disease to which the neurodegenerative disease preventive and therapeutic agent of the present invention is applied is the aforementioned neurodegenerative disease. Dementia caused by a neurodegenerative disease can also be a target disease.

As used herein, the term “dementia” refers to a result of damage to acquired brain neurons caused by a neurodegenerative disease such as Alzheimer's disease, Lewy body dementia, Parkinson's disease, or frontotemporal lobar degeneration. It is a symptom that once normal development of mental function declines, it becomes impossible to carry out daily life and social life. In addition, cerebrovascular dementia is not a subject of this specification.

Although any disease may be of any severity, it is preferably mild, that is, at an early stage, and a therapeutic effect after administration of a neurodegenerative disease preventive or therapeutic agent can be expected. In addition, in the case of a hereditary neurodegenerative disease such as Huntington's disease, a person who has (or has) a patient as a relative can be an application target regardless of whether or not it has developed.

The synapse formation enhancer, which is an active ingredient of a neurodegenerative disease preventive and therapeutic agent, has almost no side effects on the living body. Therefore, the prophylactic and therapeutic agent for neurodegenerative diseases of the present invention can be applied to healthy subjects who do not suffer from the above diseases.

2-5. Effect The preventive or therapeutic agent for neurodegenerative diseases of the present invention can prevent the onset of motor dysfunction and dementia due to neurodegenerative diseases, and can reduce or improve the symptoms. Furthermore, the neurodegenerative disease preventive and therapeutic agent of the present invention does not directly target the pathogenicity gene or its product causing the neurodegenerative disease, but by constructing a strong neural network by enhancing synapse formation, Enhance resistance to gene product toxicity. That is, since neurodegenerative diseases can be treated without treating pathogenicity genes and their products, it can be widely applied not only to specific neurodegenerative diseases but also to neurodegenerative diseases that involve synapse destruction and disappearance.

Hereinafter, the present invention will be described with specific examples. However, the following examples are merely illustrative of the present invention, and the conditions of the present invention are not limited to the conditions described in the examples. The basic experimental method in each example follows the method described in Green, MR and Sambrook, J., 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. It was.

<Example 1: miR-132 and miR-212 expression profiles in the brain of Huntington's disease model mice>
(the purpose)
Regarding miR-132 and miR-212 that constitute the synapse formation enhancer of the present invention, the expression profiles in each region of the brain of normal mice and neurodegenerative disease model mice are compared and verified.

(Method)
Huntington's disease model mouse R6 / 2 strain (Mangiarini, L., et al., 1996, Cell 87, 493-506) was used as a model mouse for neurodegenerative diseases. R6 / 2 mice were obtained from the Jackson Laboratory and maintained as described by Mangiarini et al. (1996; supra). Wild type ICR strain mice were purchased from CLEA Japan.

For wild-type mice and R6 / 2 mice at 2, 7, 14, 21, 28, 35, and 56 days after birth, the brains were removed from each of the 4 mice, and 5 brain subregions (cerebral cortex, striatum) , Hippocampus, midbrain, cerebellum), and the expression levels of miR-132, miR-212, miR-16 and U6 snoRNA were examined.

Total RNA was prepared from each brain subregion using TRIzol (Thermo Fisher Scientific) according to the attached instructions. Subsequently, using the prepared total RNA as a template, using TaqMan (registered trademark) Universal PCR Master Master Mix (Thermo Fisher Fisher Scientific) and TaqMan (registered trademark) Micro RNA Assays (Thermo Fisher Fisher Scientific), the attached instructions RT-real-time PCR was performed by AB7300 Real Time PCR System (Thermo Fisher Scientific). The TaqMan® MicroRNA Assays used and their assay IDs (indicated in parentheses) are as follows: miR-16 (000391), hsa-miR132a (000457), hsa-miR212 (000515), U6 snoRNA (001973). The analysis was performed by the delta Ct method using the expression level of U6 snoRNA as a control. The miRNA expression level on day 2 of wild-type mice was normalized as 1.

(result)
The results are shown in FIG. Our previous study revealed that miR-132 and miR-212 expression increased dramatically in each subregion of the brain and maintained in normal mice within the first month of life (Eda, A., et al., 2011, Gene 485, 46-52). Also in this example, the result was reconfirmed. On the other hand, the expression of miR-132 and miR-212 in R6 / 2 mice once increased dramatically immediately after birth, but after reaching a plateau, it was significantly maintained without being maintained as in normal mice. Diminished. Negative control miR-16 expression was not significantly different between wild-type and R6 / 2 mice.

This result suggests that the pathogenesis of Huntington's disease is related not only to abnormalities of Huntingtin protein but also to deficiencies in miR-132 and miR-212 in the brain.

<Example 2: Compensation experiment of miR-132 in the brain of Huntington's disease model mouse>
(the purpose)
To verify whether compensating miR-132 and / or miR-212 in R6 / 2 mice improves Huntington's disease symptoms.

(Method)
(1) Construction of miR-132 expression vector In each of the aforementioned encephalopathy areas, the expression level of miR-132 is significantly higher than the expression level of miR-212 (data not shown). Therefore, in this example, miR-132 was selected for compensation experiments, and an miR-132 expression vector was constructed.

(Construction of synthetic double-stranded oligo DNA)
Oligo DNA having the base sequences shown in SEQ ID NOs: 7 and 8 was commissioned and synthesized by Sigma-Aldrich. Each single-stranded oligo DNA was annealed by a conventional method to prepare a synthetic double-stranded oligo DNA containing a gene encoding miR-132.

(Construction of miR-132 expression plasmid)
Synthetic double-stranded oligo DNA encoding miR-132 is attached to pcDNA ^TM 6.2-GW / EmGFP-miR expression vector using BLOCK-iT ^TM Pol II miR RNAi Expression Vector Kit with EmGFP (Thermo Fisher Scientific) The resulting expression plasmid was designated “pMiR-132”. pMiR-132 contains the miR-132 gene within the 3 ′ UTR of the GFP reporter gene. Further, the ^{pcDNA TM 6.2-GW / EmGFP-} miR in the accompanying ^{pcDNA TM 6.2-GW / EmGFP-} miR-neg control plasmid as a negative control, which was used as a "pMIR-Neg".

(Construction of miR-132 expression virus)
A GFP reporter gene containing the miR-132 gene in pMiR-132 and the miR-neg gene in pMiR-Neg was converted into PrimeSTAR HS DNA Polymerase with GC buffer (TAKARA BIO) using the primer pairs shown in SEQ ID NOs: 9 and 10. And amplified by PCR. Amplified products were separated by agarose gel electrophoresis and then purified using PCR & Gel purification kit (BEX) according to the attached instructions. The purified amplification product was ligated to pW-CAG-eGFP-WPRE treated with EcoRI and BamHI using the In-Fusion HD Cloning Kit (TAKARA BIO) according to the attached instructions. pW-CAG-eGFP-WPRE including miR-132 and miR-Neg, respectively, was recombined according to the method of Okada et al. (Okada, T., et al., 2009, Hum Gene Ther 20, 1013-1021). Packed in adeno-associated virus (rAAV-9). The obtained miR-132-expressing AAV9 was designated as “AAV9_miR-132”, and miR-Neg-expressing AAV9 was designated as “AAV9_miR-Neg”.

(2) Introduction of expression virus into each mouse Anesthetize 3 week-old wild type mice and R6 / 2 mice with 50 mg / kg bw somnopentyl, and then add 2 μL of AAV9_miR-132 or AAV9_miR-Neg to Hamilton Neuros. Using Syringe (HAMILTON), it was introduced stereotactically on both sides of the striatum (= ˜1 mm anterior to bregma, = ˜2 mm lateral to the midline, = ˜3 mm ventral to the skull surface).

Thereafter, the expression of miR-132 in the striatum was examined in the same manner as in Example 1.

(result)
The results are shown in FIG. R6 / 2 mice (HD) introduced with AAV9_miR-132 have a statistically significant increase in miR-132 expression compared to R6 / 2 mice introduced with AAV9_miR-Neg, and AAV9_miR-Neg is introduced. It was shown to be compensated to the same extent as wild type mice (WT). On the other hand, in the wild-type mice introduced with AAV9_miR-132, the expression level of miR-132 was excessive, but no abnormality was observed in the individual (data not shown), and miR-132 was overexpressed in neurons. It was shown that there are almost no side effects.

<Example 3: Verification of motor function test>
(the purpose)
To verify the improvement of motor function in R6 / 2 mice by miR-132 supplementation.

(Method)
The method for introducing AAV9_miR-132 or AAV9_miR-Neg into wild-type mice and R6 / 2 mice was as described in Example 2. For the motor function test, a well-known rotarod test (rotarod test) and an open field test (open field test) frequently used in the motor function test of mice were performed.

(Rotarod test)
The rotarod test is a test for measuring the cooperative movement and balance of mice. In this example, the rotarod test was carried out using Ugo Basile Rota-Rod 47600 (Ugo Basile) on 11-week-old mice 8 weeks after introduction of the viral vector. The device was programmed to accelerate the rotation of the rod from 4 rpm to 40 rpm in 300 seconds, and the time from when the mouse was placed on the rotating rod of the device to falling off the rod or rotating with the rod was recorded. . From 14:00 to 19:00, we tried 3 times a day.

(Open field test)
The open field test is a test for measuring the emotionality of a mouse. In this example, the spontaneous movement of the mouse was examined using this test. One mouse was placed in a 40 cm × 28 cm × 31 cm monitor box (SUPERMEX; Muromachi Kikai), and the self-issue of the mouse in the box was measured for 15 minutes. A behavior sensor (Pyroelectric sensor PYS-001; Muromachi Kikai) was provided at the top of the box, and the behavior of the mouse was monitored with this sensor. The test was conducted between 14:00 and 19:00. The obtained data was analyzed with Data Collection Program CompACT AMS Ver.3 (Muromachi Kikai).

(result)
FIG. 3 shows the results of the rotarod test, and FIG. 4 shows the results of the open field test. R6 / 2 mice, which are fulminant Huntington's disease model mice, are known to exhibit severe coordination failure and a sudden decline in spontaneous movement (Carter, RJ, et al., 1999, J Neurosci 19, 3248-3257; Mangiarini, L., et al., 1996, Cell 87, 493-506). As shown in FIGS. 3 and 4, in R6 / 2 mice introduced with AAV9_miR-132, the coordination and spontaneous movements were significantly improved. These results suggest that miR-132 supplementation can improve motor function in Huntington's disease patients.

<Example 4: Verification of survival test>
(the purpose)
R6 / 2 mice, which are fulminant Huntington's disease model mice, are short-lived due to a marked decline in motor function and can usually survive only about 120 days. Therefore, we will verify whether miR-132 supplementation will extend the life of R6 / 2 mice.

(Method)
The method of introducing AAV9_miR-132 or AAV9_miR-Neg into R6 / 2 mice and wild type mice was in accordance with the description in Example 2. Introducing AV9_miR-132 in 14 3-week-old R6 / 2 mice and AAV9_miR-Neg in 13 mice, and AV9_miR-132 in 14 wild-type mice and AAV9_miR-Neg in 16 mice as controls Introduced. Until all R6 / 2 mice administered with AAV9_miR-132 or AAV9_miR-Neg died, they were fed for about 150 days after birth, and the survival rate in each group during that period was calculated almost daily. For the survival test, Kaplan-Meier survival analysis was used.

(result)
The results are shown in FIG. When AV9_miR-132 was introduced into R6 / 2 mice, a significant increase in lifespan was seen compared to the control AAV9_miR-Neg. This result suggests that miR-132 supplementation can prolong Huntington's disease patients, who are generally considered to have a short life span.

<Example 5: Verification of synapse-related protein>
(the purpose)
Since the motor function and life span of R6 / 2 mice were improved by supplementing miR-132 of the present invention, the recovery effect of synapse formation-related protein in R6 / 2 mice was verified.

(Method)
(1) Preparation of mouse primary neuronal cultured cells Isolate the brain tissue of E17 embryos in the ICR strain, and add 0.5 mg trypsin-EDTA solution containing 0.1 mg / mL DNase I (Roche Diagnostics) and 5 mg / mL glucose. And treated at 37 ° C. for 20 minutes. The cells were dissociated by pipetting several times and then passed through a 70 μm nylon filter (DB). Dissociated neurons are seeded onto a culture plate coated with poly-L-lysine (Sigma-Aldrich) at a cell density of 4 × 10 ³ cells / mm ² , 1% FBS (Thermo Fisher Scientific), 2% B27 supplement (Thermo Fisher Scientific), Neurobasal medium (Thermo Fisher Scientific) supplemented with 1 mM glutamine (Sigma-Aldrich) and 10 μM 2-mercaptoethanol (Sigma-Aldrich) at 37 ° C. under 5% CO ₂ In culture. For the separation of astrocytes, the cells after 1 week in culture were trypsinized and seeded on a normal tissue culture plate at a cell density of 4 × 10 ³ cells / mm ² and subcultured. The culture plate was DMEM (Thermo Fisher) supplemented with 10% FBS, 110 mg / L sodium pyruvate (Thermo Fisher Scientific) and 1 × antibiotic (25 mg / L streptomycin and 50 U / mL penicillin; Wako). Scientific). Cells were cultured and subcultured at 37 ° C. under 5% CO ₂ .

(2) Transfection Transfection of the expression plasmid pMiR-132 or pMiR-Neg into primary neuronal cultured cells was performed using Lipofectamine (registered trademark) 2000 Reagent (Thermo Fisher Scientific) according to the instruction manual. Prior to transfection, the medium was replaced with a serum-free medium (Neurobasal medium supplemented with 2% B27 supplement, 1 mM glutamine, and 10 μM 2-mercaptoethanol), and cultured at 37 ° C. for 2 hours under 5% CO ₂ . After the culture, each expression plasmid and Lipofectamine 2000 mixture were added to the cells, and 4 hours after transfection, the medium was replaced with a complete medium containing freshly prepared fresh serum.

(3) SDS-PAGE and Western blotting The cultured cells after transfection are treated with a lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM) containing 1 × protease inhibitor cocktail (Protease Inhibitor Cocktail Tablets; Roche Diagnostics). EGTA, 1% Triton X-100). The protein concentration was measured using a protein quantification kit (DOJINDO) according to the instruction manual, and about 20 μg of protein was added to 4 times the amount of sample buffer (0.25 M Tris-HCl, 40% glycerol, 8% SDS). , 0.04% bromophenol blue, and 8% β-mercaptoethanol), boiled for 5 minutes, and separated by SDS-PAGE on a 10% polyacrylamide gel. After separation, the gel was electrically blotted onto a PVDF (polyvinylidene fluoride) membrane (Immobilon P; Millipore). The membrane after blotting was treated with a blocking solution (5% BSA (Sigma-Aldrich) /0.1% Tween-20 / TBS) for 1 hour, diluted primary antibody described below was added and incubated overnight at 4 ° C. Thereafter, the membrane was washed with 0.1% Tween-20 / TBS, and horseradish peroxidase (HRP) -labeled goat anti-mouse IgG antibody (Sigma-Aldrich) diluted with 1/5000 or goat anti-rabbit IgG antibody (Sigma -Aldrich) for 1 hour at room temperature. The antigen-antibody complex after the reaction was visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore).

As synapse formation-related proteins, post-synaptic density 95 kDa (PSD95), synapsin I (SynI), and vesicular glutamine transport protein 1 (vGluT1) were selected (Candiracci C, Barbaresi P. et al., 2007, Neuroscience) 146 (4): 1829-1840; Manca P., et al., 2014, Neuroscience, 266: 102-115). The diluted primary antibody and the dilution ratio thereof are as follows. Anti-PSD95 antibody: 1/1000 diluted mouse monoclonal [6G6-1C9] (Abcam), anti-SynI antibody: 1/1000 diluted rabbit polyclonal antibody (Abcam), anti-vGluT1 antibody: 1/1000 diluted mouse Monoclonal antibody [MAB5502] (Millipore), anti-synaptophysin (Syp) antibody: 1/1000 diluted mouse monoclonal antibody [SY38] (Dako), anti-α tubulin antibody: 1/1000 diluted mouse monoclonal antibody (Sigma- Aldrich). Syp is a synaptic vesicle membrane protein that is expressed in nerve cells and is involved in neurotransmission. Alpha tubulin was used as an internal control for each sample.

(result)
The results are shown in FIG. In R6 / 2 mice, vGluT1, SynI, and PSD95 are significantly reduced compared to those in wild type mice. However, in R6 / 2 mice (HD_miR-132) introduced with AAV9_miR-132, these proteins recovered to almost the same level as wild-type mice. This result suggests that supplementation with miR-132 of the present invention can improve abnormal synapse formation in Huntington's disease patients.

<Example 6: Verification of synapse formation>
(the purpose)
Huntingtin (hereinafter referred to as “HTT”) is a causative protein of Huntington's disease, and is caused by intracellular accumulation of an abnormal HTT gene product in which the CAG repeat sequence in the HTT gene is longer than the normal HTT gene. When wild-type HTT and abnormal HTT expression plasmids were introduced into primary neuronal culture cells together with miR-132 or miR-Neg expression plasmids, the inhibition of synapse formation caused by abnormal HTT was co-expressed. Whether it can be suppressed by 132 is verified in vitro.

(Method)
(1) Construction of huntingtin expression plasmid The plasmids pEGFP-Q22 and pEGFP-Q145 constructed by Wanzhao et al. Were used as the HTT expression plasmid (Wanzhao L., et al., 2003, Proc Japan Acad 79, 293-229 / 298). pEGFP-Q22 and pEGFP-Q145 are fusion genes in which the first exon of the HTT gene containing 22 normal CAG repeat sequences and 145 abnormal CAG repeat sequences is linked to the EGFP reporter gene. PDsRed-Q-Wt and pDsRed-Q-Mt were newly constructed from each of the plasmids. pDsRed-Q-Wt and pDsRed-Q-Mt are obtained by amplifying the first exon region of the HTT gene by PCR using the primer pair shown in SEQ ID NOs: 11 and 12, and the amplified product is expressed by pDsRed-monomer vector (TAKARA BIO This is a fusion gene linked to the DsRed-monomer gene derived from

(2) Preparation of mouse primary neuronal culture cells The method described in Example 5 was followed. Wild-type ICR strains were used for mice.

(3) Transfection pEGFP-Q22 or pEGFP-Q145 was introduced together with pMiR-132 and pMiR-Neg. The transfection method was in accordance with the method described in Example 5.

(4) Immunocytochemical staining Two days after transfection, the cells were washed with PBS buffer, fixed with 4% paraformaldehyde (PFA) / PBS, and then 0.2% Triton-X / PBS at room temperature for 5 minutes. After permeabilization, the cells were incubated with a blocking solution (5% BSA (Sigma-Aldrich) / 5% goat serum (Cell signaling technology)) at room temperature for 1 hour. Synapse formation in the treated cells was detected using an anti-SynI antibody. The diluted primary antibody described below was added and incubated overnight at 4 ° C. Subsequently, the plate was washed with PBS, and the antigen-antibody complex was visualized with an isotype-specific secondary antibody labeled with Alexa-488 or Alexa-594 (both from Molecular Probes). In addition, nuclear staining was performed with 2 μg / mL Hoechst33342 (Cell signaling Technology) / PBS or 2 μg / mL propidium iodide (PI) (Thermo Fisher Scientific) / PBS. The stained cells were observed using a ZEISS fluorescence microscope (Axiovert 40 CFL).

The diluted primary antibody and the dilution ratio thereof are as follows: anti-SynI antibody is 1/1000 diluted rabbit polyclonal antibody (Abcam); anti-HTT antibody is 1/200 diluted mouse monoclonal antibody [MAB5374] ( The anti-GFP antibody used was rabbit polyclonal antibody [A11122] (Thermo Fisher Fisher Scientific) diluted 1/500.

Ten GFP-positive neurites expressing the HTT gene were arbitrarily selected, and the number of SynI-positive synaptic spines on the neurite per 10 μm was counted.

(result)
The results are shown in FIG. The number of SynI-positive synaptic spines on neurites is pEGFP-Q145 and pMiR-Neg expressing normal HTT in cells transfected with pEGFP-Q145 expressing mutant HTT and pMiR-Neg, a negative control miRNA. However, in cells transfected with pEGFP-Q145 and pMiR-132, cells with pEGFP-Q22 and pMiR-132 were introduced even though they were under the expression of mutant HTT. There was no significant decrease compared to. This result strongly suggests that if miR-132 is supplemented, inhibition of synapse formation by mutant HTT can be avoided.

<Example 7: Verification of expression suppression of mutant HTT by miR-132>
(the purpose)
Huntington's disease is caused by the accumulation of abnormal HTT inclusions in nerve cells. It is verified whether miR-132 suppresses inhibition of synapse formation in the presence of abnormal HTT due to suppression of abnormal HTT expression by miR-132.

(Method)
Mutant HTT inclusions accumulated in brain neurons of Huntington model mice were detected by immunohistochemistry. AAV9_miR-132 or AAV9_miR-Neg was administered to R6 / 2 mice in the same manner as in Example 2. At 4 weeks after administration, brains were removed from the mice by dissection and fixed with 4% PFA / PBS at 4 ° C. overnight. Next, after being immersed in 20% sucrose / PBS, it was again incubated overnight at 4 ° C. Thereafter, the brain specimen was frozen and embedded in an OCT compound (Sakura Finetech) in a dry ice / ethanol bath and cut into a sagittal section to produce a 15 μm thick cryosection. Subsequently, the cryosection was treated with blocking / permeation buffer (0.3% Triton X-100 / 5% goat serum / PBS) for 30 minutes at room temperature and washed with PBS containing 0.1% Triton X-100 and 5% goat serum. Diluted primary antibody was added and incubated overnight at 4 ° C. After washing with PBS, the sections were incubated with an isotype-specific secondary antibody labeled with Alexa-488 or Alexa-594 (both from Molecular Probes) and used with a Leica confocal fluorescence microscope (Leica TCS SP2). And observed. Nuclei were stained with 2 μg / mL Hoechst33342 (Cell signaling Technology) / PBS. The diluted primary antibody and its dilution ratio are as follows: anti-HTT antibody is 1/200 diluted mouse monoclonal antibody [MAB5374] (Millipore), anti-GFP antibody is 1/500 diluted rabbit polyclonal antibody [A11122] (Thermo Fisher Scientific) was used.

(result)
The results are shown in FIG. A is a figure which shows the result of the brain immunohistochemistry in a R6 / 2 mouse | mouth. In the figure, arrows indicate inclusion bodies observed in GFP positive cells. B shows the number of GFP positive cells including inclusion bodies in 3 different fields per mouse (247 μm × 247 μm / field).

There was no significant difference in the number of inclusion bodies between the neurons of mice administered with AAV9_miR-132 or AAV9_miR-NegR6 / 2.

In addition, the expression level of the mutant HTT transgene in R6 / 2 mice treated with AAV9_miR-132 or AAV9_miR-Neg was confirmed by RT-PCR, but no change was observed (FIG. 9). These results suggest that miR-132 has little effect on the suppression of mutant HTT expression and its inclusion body formation. This means that the symptoms of Huntington's disease have improved despite inclusion of abnormal HTT inclusions in neurons. In other words, miR-132 does not target the abnormal protein that is the direct cause of the disease, even if abnormal protein such as mutant HTT accumulates in the nerve cell, and it is sufficient neural circuit by enhancing synapse formation It is thought that tolerance to toxicity is enhanced by forming a net and building a stronger brain. Therefore, miR-132 / miR-212 suggests that it is effective not only in Huntington's disease but also in the prevention and improvement of other neurodegenerative diseases such as Alzheimer's disease where synapses are destroyed and disappeared .

Summarizing the results of Examples 3-6 above, supplementation with miR-132 enhances synapse formation in the brain of Huntington's disease model mice and restores the brain to a morphologically normal state, whereby the Huntington's disease model It strongly suggests improvement of motor function and life span in mice.

<Example 8: miR-132 expression profile in cerebellum of spinocerebellar degeneration model mouse>
(the purpose)
In Examples 1 to 7, it was confirmed that miR-132 expression level decreased in each region of the brain in Huntington's disease, and that the disease was improved by supplementing miR-132 level. In this example, the expression profiles of normal mice and spinocerebellar degeneration model mice are compared and verified in order to confirm that miR-132 expression level in the brain is decreased in neurodegenerative diseases other than Huntington's disease.

(Method)
As a model mouse for spinocerebellar degeneration, autosomal dominant mutation spinal cerebellar degeneration type 1 (SCA1) model mouse Sca1 ^{154Q / 2Q} strain (The Jackson Laboratory) was used. Wild-type mice were normal littermate mice.

Brains were removed from 2 wild type mice and 3 Sca1 ^{154Q / 2Q} mice ^aged 16 to 25 weeks, and the expression level of miR-132 in the cerebellum where the most remarkable symptoms were observed in SCA1 was examined. The specific method was in accordance with the method described in Example 1.

(result)
FIG. 10 shows the results of miR-132 expression levels in the cerebellum. In the SCA1 model mouse, the expression level of miR-132 in the cerebellum was significantly different from that in the wild type mouse as in Huntington's disease.

<Example 9: miR-132 expression profile in other brain regions of spinocerebellar degeneration model mice>
(the purpose)
In Example 8, a decrease in miR-132 expression level in the cerebellum of SCA1 model mice was confirmed. In this example, the expression profile of miR-132 in other brain regions of SCA1 is verified.

(Method)
Brains were removed from 6 wild-type mice and 4 Sca1 ^{154Q / 2Q} mice at 12 weeks of age, and the expression levels of miR-132 in the cerebral cortex, midbrain, and striatum were examined. The basic method was in accordance with Examples 1 and 8.

(result)
FIG. 11 shows the results of miR-132 expression levels in the above brain regions. In the SCA1 model mouse, it was confirmed that the expression level of miR-132 was significantly reduced in the brain region other than in the cerebellum as compared with the wild type mouse.

The results of Examples 8 and 9 suggest that deficiency of miR-132 in the brain may be common not only in Huntington's disease but also in other neurodegenerative diseases such as spinocerebellar degeneration. At the same time, as with the results of Huntington's disease, there is a possibility that other neurodegenerative diseases such as spinocerebellar degeneration can improve the abnormality in the patient by supplementing miR-132 in the nerve cells. It suggests.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (4)

A synapse formation enhancer comprising a polynucleotide comprising a base sequence shown in the following (a) to (c) or a transcription product thereof.
(A) the nucleotide sequence represented by SEQ ID NO: 1 or 5,
(B) a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1 or 5, or (c) 95% or more of the base sequence represented by SEQ ID NO: 1 or 5 Nucleotide sequences having the same base identity The synapse formation enhancer according to claim 1, wherein the polynucleotide is included in a state that can be expressed in an expression vector. A neurodegenerative disease preventive or therapeutic agent comprising the synapse formation enhancer according to claim 1 or 2 as an active ingredient. The neurodegenerative disease is Huntington's disease, corticobasal degeneration, progressive supranuclear paralysis, Alzheimer's disease, Lewy body dementia, frontotemporal lobar degeneration, spinocerebellar degeneration, Parkinson's disease, or muscle atrophy The therapeutic agent for neurodegenerative diseases according to claim 3, which is lateral sclerosis.

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