Composition for inhibiting accumulation of α-synuclein, and use therefor

Abstract

One embodiment of the present disclosure is a composition for inhibiting the accumulation of α-synuclein, the composition containing an α-synuclein deletion mutant or a nucleic acid construct that expresses the deletion mutant.

Description

Composition for inhibiting alpha-synuclein accumulation and use thereof

[0001] The present invention relates to a composition for inhibiting the accumulation of alpha-synuclein and its use, and more specifically to a deletion mutant of alpha-synuclein, a nucleic acid construct encoding the deletion mutant, a composition for inhibiting the accumulation of alpha-synuclein containing these, and its use.

[0002] In the brains of patients with many dementias and neurodegenerative diseases, intracellular aggregates that characterize the disease appear in neurons and glial cells. The main constituent proteins of intracellular aggregates vary depending on the disease, and tau has been identified as the main constituent protein of the intracellular aggregates characteristic of each disease in Alzheimer's disease (AD), while α-synuclein (aS) has been identified as the main constituent protein of the intracellular aggregates characteristic of each disease in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Hereinafter, intracellular aggregates are also referred to as intracellular protein aggregates or simply protein aggregates.

[0003] It has previously been shown pathologically that protein aggregates spread throughout the brain of patients over time, and it has been reported that the progression of this abnormal pathology correlates with clinical symptoms and disease progression.

[0004] Sengoku R, Saito Y, Ikemura M, et al. Incidence and extent of Lewy body-related alpha-synucleinopathy in aging human olfactory bulb. J Neuropathol Exp Neurol 2008;67:1072-1083. Hawkes CH, Del Tredici K, Braak H. Parkinson's disease: a dual-hit hypothesis. Neuropathol Appl Neurobiol 2007;33:599-614.Goedert M, Clavaguera F, Tolnay M. The propagation of prion-like protein inclusions in neurodegenerative diseases. Trends Neurosci. 2010 Jul;33(7):317-25.

[0005] In recent years, it has been suggested that these protein aggregates have similar properties to the abnormal prion protein in prion diseases and spread from cell to cell. Specifically, it has been shown that these protein aggregates spread from cell to cell and function as seeds that trigger new aggregation within the cells they reach, resulting in the aggregation of normal proteins that would not normally accumulate within cells (e.g., α-synuclein (aS)). At the same time, it has been suggested that this cell-to-cell spread of protein aggregates and the intracellular accumulation of seed-dependent protein aggregates may be one of the mechanisms underlying the pathogenesis of these diseases.

[0006] Although research into the seed-dependent intracellular accumulation of protein aggregates is still in its infancy, finding a way to inhibit this intracellular accumulation of protein aggregates may lead to the treatment and prevention of related diseases.

[0007] In view of the above-mentioned problems, one aspect of the present invention aims to provide a composition for inhibiting alpha-synuclein accumulation and use thereof.

[0008] In order to solve the above problems, the present invention includes, for example, the following aspects.

[0009] 1) A composition for inhibiting alpha-synuclein accumulation, comprising a deletion mutant of alpha-synuclein or a nucleic acid construct expressing the deletion mutant. 2) A deletion mutant of alpha-synuclein, in which a stretch of 3 to 30 consecutive amino acids is deleted, the deletion of the consecutive amino acids being in a region corresponding to amino acids 21 to 70 or to amino acids 81 to 120 in the amino acid sequence of alpha-synuclein shown in SEQ ID NO: 1, and in which at least amino acids corresponding to amino acids 43 and / or 44, or at least amino acids corresponding to amino acids 103 and / or 104 in the amino acid sequence of SEQ ID NO: 1 are deleted. 3) A nucleic acid construct encoding the deletion mutant described in 2) above. 4) A method for inhibiting seed-dependent alpha-synuclein accumulation, comprising a step of coexisting a deletion mutant of alpha-synuclein with wild-type alpha-synuclein. 5) A method for screening a substance that inhibits seed-dependent alpha-synuclein accumulation, comprising a step of detecting whether aggregation of wild-type alpha-synuclein is inhibited in the coexistence of wild-type alpha-synuclein, a candidate substance, and alpha-synuclein seeds.

[0010] According to one aspect of the present invention, a composition for inhibiting alpha-synuclein accumulation and use thereof can be provided.

[0011] Figure 1 shows the primary structure of α-synuclein (aS). aS is a single-chain polypeptide consisting of 140 amino acids. The N-terminus contains a six-residue repeat sequence based on the KTKEGV base. The central part of the molecule contains a relatively hydrophobic region called NAC. The C-terminus contains a phosphorylation site (Ser at residue 129). Genetic analysis of patients with familial Parkinson's disease and dementia with Lewy bodies has revealed several missense mutations (e.g., A30P and A53T). Figure 2 shows the seed-dependent accumulation of recombinant aS monomer, an example of the present invention. Monomers (1 mg / mL) of wild-type (WT), mutants lacking 31-40 amino acid residues (d31-40), d41-50 lacking 41-50 amino acid residues, d51-60 lacking 51-60 amino acid residues, and d61-70 lacking 61-70 amino acid residues were mixed with WT aggregates (WT seeds) and incubated at 37°C in the presence of thioflavin T (ThT). The ThT fluorescence intensity of these samples was continuously measured. While a seed-dependent increase in ThT fluorescence intensity was observed for the WT monomer, no seed-dependent increase in ThT was observed for any of the mutant monomers. Figure 3 shows the results of RP-HPLC analysis of recombinant aS protein, an example of the present invention. Recombinant proteins of wild-type (WT), mutants with deletions of amino acid residues 31-40 (d31-40), mutants with deletions of amino acid residues 41-50 (d41-50), mutants with deletions of amino acid residues 51-60 (d51-60), and mutants with deletions of amino acid residues 61-70 (d61-70) were analyzed by RP-HPLC. WT eluted at 7.580 minutes, while the other mutants eluted at different retention times from WT. These results demonstrated that each recombinant aS had a different retention time under these RP-HPLC analysis conditions. Figure 4 shows the effect of aS deletion mutants on seed-dependent WT accumulation, an example of the present invention. WT and deletion mutant aS were mixed, and WT seeds were added. After incubation at 37°C for 2 days, the accumulated aS was collected by centrifugation. The precipitated fraction was analyzed by RP-HPLC, and the accumulated WT and deletion mutant aS were quantified.When the accumulation level in WT + saline with WT seeds was taken as 100%, the seed-dependent accumulation of WT was suppressed by approximately 70% in WT + d41-50 with WT seeds. Figure 5 shows the inhibitory effect of d41-50 on seed-dependent WT accumulation, an example of the present invention. 50 μL of WT, d41-50 monomer (2 mg / mL), or 50 μL of saline (saline) was added to 50 μL of WT monomer (2 mg / mL), and then thioflavin T (ThT) and 2 μg of WT seeds were mixed. Each sample was incubated at 37°C, and the ThT fluorescence intensity was continuously measured at an excitation wavelength of 442 nm and an emission wavelength of 485 nm. Figure 6 shows the effect of deletion mutants on seed-dependent WT accumulation in cultured cells, an example of the present invention. WT and deletion mutant aS were transiently expressed in SH-SY5Y cells. The WT seed was then introduced into the cells and incubated at 37°C for 3 days. The cells were harvested, and sarkosyl-soluble (sup) and insoluble (ppt) fractions were prepared and subjected to immunoblotting. The upper panel detected aS using an antibody recognizing the 131-140 amino acid region of aS (anti-131-140), while the lower panel detected aS using a phosphorylated antibody recognizing Ser129 of aS (anti-64). In the figure, 1-6 refer to the following: 1: WT + empty vector (ev), 2: WT + ev + WT seed (seed), 3: WT + WT + seed, 4: WT + d21-30 + seed, 5: WT + d31-40 + seed, and 6: WT + d41-50 + seed. Figure 7 shows a comparison of the effects of deletion mutants on seed-dependent WT accumulation in cultured cells, an example of the present invention. The band abundance in the sarkosyl-insoluble fraction (ppt) of cells expressing WT, empty vector (empty), or each deletion mutant was quantified after seeding. The band abundance in cells expressing each deletion mutant was shown relative to the band abundance in cells expressing empty vector (100%). Figure 8 shows the effect of deletion mutants on seed-dependent WT accumulation in cultured cells, an example of the present invention. WT and deletion mutant aS were transiently expressed in SH-SY5Y cells.The WT seed was then introduced into the cells and incubated at 37°C for 5 days. The cells were harvested, and sarkosyl-soluble (sup) and insoluble (ppt) fractions were prepared and immunoblotted. The upper panel detected aS using an antibody recognizing the 131-140 amino acid region of aS (anti-131-140), while the lower panel detected aS using a phosphorylated antibody recognizing Ser129 of aS (anti-64). Numbers 1 to 6 in the figure refer to the following: 1: WT + empty vector (ev), 2: WT + ev + WT seed (seed), 3: WT + d43-45 + seed, 4: WT + d43-46 + seed, 5: WT + d43-48 + seed, and 6: WT + d41-50 + seed. Figure 9 shows the effect of deletion mutants on MSA patient brain seed (MSA seed)-dependent WT accumulation, an example of the present invention. WT and deletion mutant aS were transiently expressed in SH-SY5Y cells. Then, MSA seeds were introduced into the cells and incubated at 37°C for 3 days. Cells were harvested, and sarkosyl-soluble (sup) and insoluble (ppt) fractions were prepared and immunoblotted. The upper panel detected aS using an antibody recognizing the 131-140 amino acid region of aS (anti-131-140), while the lower panel detected aS using a phosphorylated antibody recognizing Ser129 (anti-64). In the figure, 1-6 refer to the following: 1: WT + empty vector (ev), 2: WT + ev + MSA seeds, 3: WT + WT + MSA seeds, 4: WT + d21-30 + MSA seeds, 5: WT + d31-40 + MSA seeds, and 6: WT + d41-50 + MSA seeds. Figure 10 shows the effect of deletion mutants on DLB seed-derived WT accumulation, an example of the present invention. WT and deletion mutant aS were transiently expressed in SH-SY5Y cells. DLB seeds were then introduced into the cells and incubated at 37°C for 3 days. Cells were harvested, and sarkosyl-soluble (sup) and insoluble (ppt) fractions were prepared and immunoblotted. The upper panel shows aS detected using an antibody (anti-131-140) recognizing the 131-140 amino acid region of aS, while the lower panel shows a phosphorylated antibody (anti-64) recognizing Ser129 of aS.In the figure, 1 to 6 refer to the following: 1: WT + empty vector (ev), 2: WT + ev + DLB seed, 3: WT + WT + DLB seed, 4: WT + d21-30 + DLB seed, 5: WT + d31-40 + DLB seed, 6: WT + d41-50 + DLB seed. Figure 11 shows the results of immunohistochemical staining of phosphorylated aS aggregates in mouse brains infected with AAV and inoculated with recombinant aS aggregates, as an example of the present invention. Recombinant aS fibers (seed) were inoculated into mouse brains preinfected with AAV-GFP expressing GFP or AAV-d41-50 expressing a deletion mutant aS. After 3 months, the brains were removed, thin sections were prepared, and stained with an anti-phosphorylated aS antibody (pS129). Photographs of staining in the striatum and cortex on the inoculated side are shown. Figure 12 shows the results of quantifying the amount of phosphorylated aS aggregates in mouse brains, an example of the present invention. Recombinant aS fibers (seed) were inoculated into mouse brains previously infected with AAV-GFP, which expresses GFP, or AAV-d41-50, which expresses a deletion mutant aS. Three months later, the brains were removed, thin sections were prepared, and stained with anti-phosphorylated aS antibody (pS129). These sections were analyzed using a BZ-X710 microscope, and the pS129-positive area was calculated. The percentage of this area was used as the amount of phosphorylated aS aggregates.

[0012] 1. Deletion Mutants of Alpha-Synuclein and Nucleic Acid Constructs Expressing These Deletion Mutants (Deletion Mutants) A deletion mutant of alpha-synuclein (aS) according to one embodiment of the present invention refers to a mutant in which some amino acids are deleted from the amino acid sequence constituting the primary structure of wild-type alpha-synuclein. This deletion mutant is also sometimes referred to as a deletion mutant type of alpha-synuclein.

[0013] The number of amino acids deleted in a deletion mutant compared with the amino acid sequence of wild-type alpha-synuclein is not particularly limited, but may be, for example, within the range of 1 to 40 amino acids. The lower limit of the number of amino acids deleted in a deletion mutant is, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more amino acids. The upper limit of the number of amino acids deleted in a deletion mutant is, for example, 40 or fewer, 30 or fewer, 25 or fewer, 20 or fewer, or 15 or fewer. The number of amino acids deleted in a deletion mutant may preferably be within the range of 3, 4, or 5 or more to 30 amino acids, 3, 4, or 5 or more to 25 amino acids, 3, 4, or 5 or more to 20 amino acids, or 3, 4, or 5 or more to 15 amino acids.

[0014] In a preferred example, the deletion of the amino acids in the deletion mutant is a deletion of consecutive amino acids. For example, the deletion mutant is missing 1 or more and 40 or less consecutive amino acids, 3, 4, or 5 or more and 30 or less consecutive amino acids, 3, 4, or 5 or more and 25 or less consecutive amino acids, 3, 4, or 5 or more and 20 or less consecutive amino acids, or 3, 4, or 5 or more and 15 or less consecutive amino acids. This deletion mutant may also be missing 6, 7, 8, 9, or 10 or more consecutive amino acids.

[0015] The deletion of consecutive amino acids in the deletion mutant is preferably a deletion in the region corresponding to the 21st to 70th amino acids, the region corresponding to the 31st to 70th amino acids, the region corresponding to the 81st to 120th amino acids, or the region corresponding to the 91st to 120th amino acids in the amino acid sequence of human α-synuclein shown in SEQ ID NO: 1. The deletion of consecutive amino acids in the deletion mutant is more preferably a deletion in the region corresponding to the 21st to 60th amino acids or the region corresponding to the 31st to 60th amino acids in the amino acid sequence of α-synuclein shown in SEQ ID NO: 1. The deletion of consecutive amino acids in the deletion mutant is even more preferably a deletion in the region corresponding to the 35th to 55th amino acids in the amino acid sequence of α-synuclein shown in SEQ ID NO: 1. The deletion of consecutive amino acids in the deletion mutant is particularly preferably a deletion in the region corresponding to the 37th, 38th, 39th, or 40th to 53rd, 52nd, 51st, or 50th amino acids in the amino acid sequence of α-synuclein shown in SEQ ID NO: 1.

[0016] A preferred example of a deletion mutant of α-synuclein combines the structural properties exemplified above, and for example, satisfies all of the following conditions 1) to 3): 1) A sequence of 3, 4, or 5 or more but not exceeding 30 consecutive amino acids is deleted. As described above, the deletion of consecutive amino acids may be a sequence of 3, 4, or 5 or more but not exceeding 25 consecutive amino acids, a sequence of 3, 4, or 5 or more but not exceeding 20 consecutive amino acids, or a sequence of 3, 4, or 5 or more but not exceeding 15 consecutive amino acids. 2) The deletion of consecutive amino acids is a deletion in the region corresponding to the 21st to 70th amino acids, the region corresponding to the 31st to 70th amino acids, the region corresponding to the 81st to 120th amino acids, or the region corresponding to the 91st to 120th amino acids in the amino acid sequence of α-synuclein shown in SEQ ID NO: 1. As described above, the region in which the consecutive amino acids are deleted may be a deletion in the region corresponding to amino acids 31 to 60, a deletion in the region corresponding to amino acids 35 to 55, or a deletion in the region corresponding to amino acids 37, 38, 39, or 40 to 53, 52, 51, or 50 in the amino acid sequence of alpha-synuclein shown in SEQ ID NO: 1. 3) At least the amino acids corresponding to positions 43 and / or 44 in the amino acid sequence shown in SEQ ID NO: 1 are deleted, or at least the amino acids corresponding to positions 103 and / or 104 are deleted. In a typical example, the deletion mutant is at least the amino acids corresponding to positions 43 and 44 in SEQ ID NO: 1, or at least the amino acids corresponding to positions 103 and 104.

[0017] As used herein, the term "amino acid residue corresponding to a specific amino acid residue in the amino acid sequence shown in SEQ ID NO: 1" refers to 1) the specific amino acid residue itself in the amino acid sequence shown in SEQ ID NO: 1, and 2) an amino acid residue in another amino acid sequence that corresponds to the specific amino acid residue in the amino acid sequence shown in SEQ ID NO: 1. In the case of 2), it refers to amino acid residue X in another amino acid sequence that is identified by homology analysis as corresponding to amino acid residue X in the amino acid sequence shown in SEQ ID NO: 1. Examples of homology analysis methods include pairwise sequence alignment methods such as the Needleman-Wunsch method and the Smith-Waterman method, and multiple sequence alignment methods such as the ClustalW method. Those skilled in the art can use these methods to determine the "corresponding amino acid residue" in another amino acid sequence to be analyzed using the amino acid sequence shown in SEQ ID NO: 1 as a reference sequence. Examples of other amino acid sequences to be analyzed include isoforms, homologs, or variants of the reference sequence. Analysis may be performed using default settings, or parameters may be changed from the defaults as needed.

[0018] In one example of an α-synuclein deletion mutant, the amino acid sequence excluding the deletion region exhibits, for example, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity when compared with the amino acid sequence set forth in SEQ ID NO: 1. The α-synuclein deletion mutant is preferably a deletion mutant based on the wild-type sequence of human α-synuclein, but may also be a deletion mutant based on the wild-type sequence of α-synuclein from other animals (preferably mammals) such as mouse, rat, or pig.

[0019] As described below, deletion mutants of alpha-synuclein can suppress, for example, seed-dependent accumulation of alpha-synuclein and can be used for the treatment or prevention of alpha-synucleinopathy.

[0020] (Nucleic acid construct expressing deletion mutant) A nucleic acid construct according to one embodiment of the present invention expresses any of the deletion mutants of α-synuclein described above. The type of nucleic acid constituting the nucleic acid construct is not particularly limited, and examples include those composed of DNA, those composed of RNA, and those composed of DNA and RNA. The nucleic acid construct may also contain a non-naturally occurring nucleic acid, as necessary.

[0021] One form of the nucleic acid construct according to this embodiment is a gene expressing any of the above-described deletion mutants of α-synuclein. The base sequence of the gene expressing a deletion mutant of α-synuclein, excluding the deletion region, exhibits, for example, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the base sequence shown in SEQ ID NO: 2 (human α-synuclein gene). The deletion mutant of α-synuclein is preferably a deletion mutant based on the wild-type sequence of human α-synuclein, but may also be a deletion mutant based on the wild-type sequence of α-synuclein from other animals (preferably mammals) such as mice, rats, or pigs.

[0022] Another form of the nucleic acid construct according to this embodiment is an expression cassette that expresses the above-mentioned gene. The expression cassette is preferably an expression vector, the type of which is appropriately selected depending on the host cell. The expression vector is more preferably a viral vector such as a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an adeno-associated viral vector, a herpes viral vector, or a vaccinia viral vector. Among these, adeno-associated viral vectors are preferred from the viewpoint of use in gene therapy for neurological diseases. Among these, adeno-associated viral vectors such as serotype 1 (AAV1), which exhibits tissue tropism for the central nervous system, serotype 2 (AAV2), which has broad tissue tropism, and serotype 5 (AAV5), which exhibits tissue tropism for the central nervous system, may be preferred from the viewpoint of use in gene therapy for neurological diseases.

[0023] The expression cassette includes any expression control region that is functional in the expression host. The type of promoter that is the expression control region is not particularly limited, but specifically, for example, promoters that bring about high expression in mammals (especially humans) or that function selectively in specific tissues, such as the CAG promoter, CMV promoter, and SYN (synapsin) I, may be preferred. Among these promoters, the CAG promoter may be more preferred. In the expression cassette, the promoter is located upstream of the gene.

[0024] The expression cassette may further include a functional sequence, if necessary, that has the effect of increasing the stability of mRNA produced by transcription and functions to improve gene expression. An example of a functional sequence is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. In the expression cassette, the WPRE sequence is located downstream of the gene.

[0025] Another example of a functional sequence that may be included in the expression cassette is a poly-A tail addition signal sequence. Examples of poly-A tail addition signal sequences include the sequence of Simian Virus 40 and the sequence of human growth hormone, with the sequence of human growth hormone being more preferred. In the expression cassette, the WPRE sequence is located downstream of the gene.

[0026] The expression cassette may further include ITRs (inverted terminal repeat sequences), etc. The ITRs are arranged in the expression cassette so as to sandwich the entire transcription unit (from the promoter to the polyA tail addition signal sequence).

[0027] [2. Composition for inhibiting alpha-synuclein accumulation] A composition according to one embodiment of the present invention comprises a deletion mutant of alpha-synuclein or a nucleic acid construct that expresses the deletion mutant. The deletion mutant of alpha-synuclein and the nucleic acid construct that expresses the deletion mutant are as described above in the section [1. Deletion mutant of alpha-synuclein and nucleic acid construct that expresses the deletion mutant].

[0028] The composition according to this embodiment can be used as a composition for inhibiting the accumulation of α-synuclein (particularly wild-type α-synuclein). This composition can be used, in particular, for the purpose of inhibiting seed-dependent α-synuclein accumulation. Here, "inhibition of α-synuclein accumulation" refers to the inhibition of α-synuclein (typically wild-type α-synuclein) accumulation compared to the case in which the composition according to this embodiment is not present. The degree of inhibition of α-synuclein accumulation is not particularly limited, but refers to, when the amount of α-synuclein accumulated in the absence of the composition according to this embodiment is taken as 100%, the amount of accumulation can be, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or substantially no accumulation. Here, the inhibition of α-synuclein accumulation can be a phenomenon occurring in an in vitro (cell-free) environment or an intracellular environment. The intracellular environment may be within an isolated cell or may be present in the body of a human or non-human animal. The cells are preferably nervous system cells, including cells of the central nervous system, cells of the peripheral nervous system, glial cells, and precursor cells of these cells.

[0029] Seed-dependent α-synuclein accumulation refers to the accumulation of α-synuclein in which abnormal α-synuclein protein molecules serve as aggregation nuclei (seeds). The abnormal α-synuclein protein molecules form aggregates. The α-synuclein aggregates may be, for example, α-synuclein aggregates contained in living organisms or isolated α-synuclein aggregates. The α-synuclein aggregates may be artificially constructed in vitro, artificially constructed in cells using techniques such as genetic recombination, or naturally generated in living organisms. For example, seed α-synuclein aggregates can be obtained by denaturing and fibrillating artificially constructed wild-type α-synuclein or in the brains of patients with α-synucleopathy (see also the Examples).

[0030] A composition according to one embodiment of the present invention is a composition for treating or preventing α-synucleinopathy, i.e., a pharmaceutical composition. The pharmaceutical composition according to one embodiment of the present invention preferably comprises the nucleic acid construct described above. As described above, this pharmaceutical composition suppresses α-synuclein accumulation, particularly seed-dependent α-synuclein accumulation, in nervous system cells present in the body of a human or non-human animal. It also suppresses the propagation of abnormally accumulated α-synuclein aggregates between nervous system cells that occurs following seed-dependent α-synuclein accumulation. As a result, prophylactic and therapeutic effects such as prevention, halting progression, or delaying progression of α-synucleinopathy are achieved. The scope of the present invention includes methods for treating and / or preventing α-synucleinopathy, in which an effective amount of this pharmaceutical composition is administered to a subject.

[0031] The human or non-human animal to which the pharmaceutical composition is administered is preferably any one selected from the group consisting of mammals including humans. The type of mammal to which the pharmaceutical composition is administered is not particularly limited, and examples include laboratory animals such as mice, rats, rabbits, guinea pigs, and primates other than humans; pet animals such as dogs and cats; livestock such as cows and horses; and humans; with humans being particularly preferred. The human or non-human animal to which the pharmaceutical composition is administered may, for example, have developed α-synucleinopathy, as described below, or be in a pre-development stage.

[0032] The type of α-synucleinopathy (also simply referred to as synucleinopathy) to be treated and / or prevented is not particularly limited, and broadly refers to neurodegenerative diseases characterized by abnormal structure and aggregation of α-synuclein in neurons and glial cells, including, for example, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA).

[0033] The method of administration of the pharmaceutical composition is not particularly limited, and may be local administration by techniques such as injection (using a syringe or an infusion pump, etc.), ophthalmic administration, transdermal administration, sublingual administration, etc., or systemic administration by techniques such as oral administration, intravenous or intraarterial administration, intestinal administration, etc. In one preferred administration mode, the pharmaceutical composition is administered locally to the vicinity of the nervous system that is the target of treatment, etc.

[0034] The dosage (effective amount) of the pharmaceutical composition may be appropriately determined depending on the age, sex, symptoms, administration route, number of administrations, etc. of the human or animal to be administered. If necessary, an in vivo assay using the pharmaceutical composition can be carried out in advance to determine the dosage without the need for excessive experimentation.

[0035] The number of times the pharmaceutical composition is administered is not particularly limited as long as the effect is obtained, and may be appropriately determined depending on, for example, the dosage, administration route, symptoms, and the age and sex of the human or animal.

[0036] A composition (including a pharmaceutical composition) according to one embodiment of the present invention may comprise at least the deletion mutant of α-synuclein or a nucleic acid construct expressing the deletion mutant, and a carrier (e.g., a pharmaceutically acceptable carrier). The carrier is not particularly limited, but preferably has the properties of not substantially inhibiting the function of the deletion mutant of α-synuclein or the nucleic acid construct and not substantially adversely affecting the human or non-human animal to which it is administered. It may also be preferable for the composition to contain a liquid carrier such as water, which may be a liposomal formulation, for example.

[0037] Further examples of components constituting the above composition include, but are not limited to, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, colorants, antioxidants, viscosity adjusters, etc.

[0038] [3. Methods for inhibiting alpha-synuclein accumulation, methods for screening for substances that inhibit accumulation, etc.] A method according to one embodiment of the present invention is a method for inhibiting seed-dependent alpha-synuclein accumulation, comprising a step of allowing a deletion mutant of alpha-synuclein to coexist with wild-type alpha-synuclein. The deletion mutant of alpha-synuclein can be provided, for example, using the composition described in the above section [2. Composition for inhibiting alpha-synuclein accumulation] or expressed from a nucleic acid construct. The step of allowing the deletion mutant of alpha-synuclein to coexist with wild-type alpha-synuclein can be performed in vitro (cell-free), intracellular, or in vivo (in the brain of experimental animals such as mice or rats). For details of the method for inhibiting alpha-synuclein accumulation, please refer to the description in the above section [2. Composition for inhibiting alpha-synuclein accumulation]. A method according to one embodiment of the present invention is a screening method for a substance that inhibits seed-dependent alpha-synuclein accumulation, and includes a step of detecting whether wild-type alpha-synuclein aggregation is inhibited in the coexistence of wild-type alpha-synuclein, a candidate substance, and alpha-synuclein seeds. If wild-type alpha-synuclein aggregation is inhibited compared to the absence of the candidate substance, the candidate substance is selected as a substance that inhibits alpha-synuclein accumulation. Note that the wild-type alpha-synuclein and alpha-synuclein seeds may be, for example, those described in the above section "2. Composition for inhibiting alpha-synuclein accumulation." Furthermore, methods for detecting whether wild-type alpha-synuclein aggregation is inhibited include, for example, 1) a method in which ThT is present in a system and detection is performed based on a change in ThT fluorescence intensity; and 2) a method in which the presence or absence and degree of wild-type alpha-synuclein aggregation is examined using HPLC or the like.

[0039] (Summary) By summarizing the above embodiments, the present invention can be summarized as follows.

[0040] 1) A composition for inhibiting alpha-synuclein accumulation, comprising a deletion mutant of alpha-synuclein or a nucleic acid construct expressing the deletion mutant. 2) The composition according to 1), wherein the deletion mutant of alpha-synuclein is deficient in a sequence of 1 to 40 consecutive amino acids. 3) The composition according to 2), wherein the deletion mutant of alpha-synuclein is deficient in a sequence of 3 to 15 consecutive amino acids. 4) The composition according to any of 1) to 3), which inhibits seed-dependent alpha-synuclein accumulation. 5) The composition according to any of 2) to 4), wherein the deletion of consecutive amino acids is in a region corresponding to amino acids 21 to 70 or to amino acids 81 to 120 in the amino acid sequence of alpha-synuclein shown in SEQ ID NO: 1. 6) The composition according to 5), wherein the deletion of consecutive amino acids is in a region corresponding to amino acids 35 to 55 in the amino acid sequence of alpha-synuclein shown in SEQ ID NO: 1. 7) The composition according to any one of 1) to 6), which is for treating or preventing α-synucleinopathy. 8) A deletion mutant of α-synuclein, which is missing 3 or more consecutive amino acids and 30 or less consecutive amino acids, the deletion of the consecutive amino acids being in a region corresponding to amino acids 21 to 70 or to amino acids 81 to 120 in the amino acid sequence of α-synuclein shown in SEQ ID NO: 1, and which is missing at least amino acids corresponding to amino acids 43 and / or 44, or at least amino acids corresponding to amino acids 103 and / or 104 in the amino acid sequence shown in SEQ ID NO: 1. 9) A nucleic acid construct encoding the deletion mutant according to 8) above. 10) A method for suppressing seed-dependent α-synuclein accumulation, which comprises a step of allowing a deletion mutant of α-synuclein to coexist with wild-type α-synuclein. 11) A method for screening a substance that inhibits seed-dependent alpha-synuclein accumulation, comprising a step of detecting whether aggregation of wild-type alpha-synuclein is inhibited in the coexistence of wild-type alpha-synuclein, a candidate substance, and alpha-synuclein seeds.

[0041] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0042] An embodiment of the present invention will now be described.

[0043] Example 1 Overview of Example 1 The inventors conducted experiments to identify a factor that inhibits the seed-dependent accumulation of aS (α-synuclein). It is believed that this factor inhibits the seed-dependent aggregation of aS by interacting with aS aggregates and / or aS monomer molecules, which act as seeds. However, since numerous factors (proteins, etc.) that interact with aS aggregates are expected to exist, the search for such a factor is extremely difficult. Therefore, the inventors focused on the aS protein itself, conceiving that its mutant form might interact with aS aggregates and inhibit their seed-dependent aggregation. They created various aS mutants and investigated their effects in vitro. As a result, they found that deletion mutant aS inhibited the seed-dependent accumulation of wild-type aS.

[0044] In vitro, the addition of wild-type aS aggregate seeds to a recombinant wild-type aS monomer solution resulted in seed-dependent aggregation of wild-type aS monomers. We found that the seed-dependent accumulation of wild-type monomers was suppressed when wild-type aS monomers were coexisted with an equal amount of deletion mutant aS monomers and wild-type aS aggregate seeds were added. Furthermore, when wild-type aS plasmid was transiently expressed in cultured cells and then wild-type aS aggregate seeds were introduced into the cells, the wild-type aS derived from the plasmid accumulated intracellularly. When both the wild-type and deletion mutant aS plasmids were transiently expressed and seeds were added, the intracellular accumulation of wild-type aS was reduced compared to cells expressing wild-type aS alone. These in vitro experimental results demonstrated that deletion mutant aS suppresses the seed-dependent accumulation of wild-type aS. These experiments suggest that deletion mutant aS may be a novel therapeutic or preventive agent for α-synucleinopathy.

[0045] <Contents and Results of Example 1> Wild-type aS (WT) is a single-chain polypeptide consisting of 140 amino acid residues as shown in SEQ ID NO: 1 (Figure 1). In this study, we prepared recombinant proteins of deletion mutants, each deleting 10 amino acid residues from the N-terminus, and compared their aggregation properties. First, we investigated the seed-dependent accumulation and aggregation of each deletion mutant monomer. WT monomers undergo fibrillation and aggregation within a few days when shaken at 37°C, but do not aggregate when left unshaken at 37°C. However, when a small amount of pre-prepared WT aggregates is added to a stationary WT monomer solution, the WT monomer aggregates. In other words, it is believed that the added WT aggregates act as aggregation nuclei (seeds), causing the WT monomer to aggregate in a seed-dependent manner. Therefore, we investigated whether the deletion mutant aS monomers (deletion mutants) prepared in this study aggregate in a seed-dependent manner. To a 100 μL solution of WT or deletion mutant aS monomers (d31-40, which lacks 31-40 amino acid residues in SEQ ID NO:1; d41-50, which lacks 41-50 amino acid residues in SEQ ID NO:1; d51-60, which lacks 51-60 amino acid residues in SEQ ID NO:1; and d61-70, which lacks 61-70 amino acid residues in SEQ ID NO:1), 2 μg of WT aggregates (WT seed) was added, and the fluorescence intensity of ThT (thioflavin T) was continuously measured. While the WT monomer showed a seed-dependent increase in ThT fluorescence intensity, none of the deletion mutant aS monomers showed any seed-dependent increase in ThT (Figure 2). These deletion mutant aS monomers therefore did not aggregate in a seed-dependent manner.

[0046] Next, we investigated whether these deletion mutant aS monomers suppressed the seed-dependent accumulation of WT monomers. 100 μL of 5 mg / mL WT monomer solution and deletion mutant aS monomer solution were mixed, and 2 μg of WT seeds was added to the mixture and incubated at 37°C for several days. After incubation, the sample was centrifuged at 163,000 g for 20 minutes, and the seed-dependently accumulated aS was collected as a precipitate. After adding 20 μL of 6 M guanidine hydrochloride to the collected fraction to dissolve the precipitate, a portion was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) to determine the protein concentration.

[0047] First, we analyzed these four deletion mutant aS monomers by RP-HPLC. The chromatograms shown in Figure 3 revealed that each mutant eluted at a different retention time from the WT monomer. Equal amounts of each deletion mutant monomer and the WT monomer were then mixed, and WT seeds were added. After 2–3 days of incubation, seed-dependent accumulation of aS was recovered as a precipitate by centrifugation. The precipitate was dissolved in guanidine hydrochloride and analyzed by RP-HPLC. The results are shown in Figure 4. The precipitate fraction contained both the deletion mutant and the WT, and they were clearly separable under these elution conditions. Specifically, the WT eluted at approximately 7.5 min, whereas d31-40 eluted at 6.9 min, d41-50 eluted at 7.68 min, d51-60 eluted at 6.4 min, and d61-70 eluted at approximately 6.0 min, all of which were separable from the WT peak. Furthermore, the amount of WT protein accumulated in a seed-dependent manner was calculated from the WT peak area in the precipitate fraction. The amount of WT accumulated in a sample containing WT and saline was defined as 100%, and the percentage of WT accumulation upon addition of each deletion mutant aS was calculated. The lowest amount of WT accumulation was observed upon addition of d41-50, and it was found that the addition of d41-50 suppressed seed-dependent accumulation of WT by approximately 70% compared to the addition of saline. On the other hand, when other deletion mutant aS were added, seed-dependent accumulation of WT was observed to be suppressed by approximately 20%. Similar experiments were also performed using deletion mutant aS d11-20, d21-30, d71-80, d81-90, d91-100, and d101-110 (all of which lack the specified 10 amino acid residues in SEQ ID NO: 1), and a general tendency for the amount of WT accumulation to decrease was observed. Among these, d21-30 showed a tendency to suppress WT seed-dependent accumulation by approximately 50%, d101-110 showed a tendency to suppress WT seed-dependent accumulation by approximately 60%, and the others generally showed a tendency to suppress WT seed-dependent accumulation by approximately 20% to 40%.

[0048] Next, we investigated the inhibitory effect of the deletion mutant aS, d41-50, on the seed-dependent accumulation of WT by a ThT fluorescence intensity assay. WT monomer was mixed with saline, WT monomer, or d41-50 monomer, and then WT seeds and ThT were added and incubated at 37°C. The ThT fluorescence intensity in the mixture was measured over time, as shown in Figure 5. The sample in which WT monomer and saline were mixed and WT seeds were added (WT+saline+seed) showed an increase in ThT over time, indicating seed-dependent accumulation of WT monomer (Figure 5, dark gray line). The sample in which WT monomer and WT monomer were mixed and seeds were added (WT+WT+seed) showed approximately twice the fluorescence intensity at the end point compared to WT+saline+seed (Figure 5, black line). On the other hand, in the sample in which WT monomer was mixed with d41-50 monomer and seeds were added (WT+d41-50+seed), the fluorescence intensity at the end point was reduced to approximately one-quarter of that of WT+saline+seed (Fig. 5, light gray line). This indicates that the addition of d41-50 monomer suppressed seed-dependent accumulation of WT monomer by approximately 75%, a result similar to that shown in Fig. 4.

[0049] Next, we examined the effect of d41-50 on seed-dependent accumulation of WT in cultured cells. WT and deletion mutants, such as d41-50, were transiently expressed in SH-SY5Y neuroblastoma cells. Recombinant WT aggregates (WT seeds) were then introduced into the cells and cultured for 3 days. The cells were harvested and homogenized in the presence of the detergent sarkosyl. The cells were separated by ultracentrifugation into sarkosyl-soluble (sup) and insoluble (ppt) fractions, each of which was analyzed by immunoblotting. When recombinant WT seeds were introduced into WT-expressing cells, the plasmid-derived WT was phosphorylated and accumulated intracellularly in a seed-dependent manner (Figure 6, bottom panel, lane 2 of the ppt fraction). The 15 kDa band represents WT, and several bands above it represent ubiquitinated WT and multimers. When the amount of the band in lane 2 in the ppt fraction (aS accumulation) was taken as 100%, the accumulation of phosphorylated WT in the ppt fraction was reduced by approximately 60% in cells transiently co-expressing d41-50 with WT (Fig. 6, bottom panel, lane 6 of the ppt fraction). On the other hand, in cells co-expressing other deletion mutants, the accumulation of WT was suppressed by a maximum of approximately 20%.

[0050] In addition, the effects of deletion mutants other than d41-50 on WT seed-dependent aS accumulation were examined in a cultured cell system. Cells were prepared using a method similar to that shown in Figure 6, separated into sup and ppt, and then analyzed by immunoblotting. Figure 7 shows a graph summarizing the accumulation of phosphorylated WT in ppt. These results demonstrate that seed-dependent intracellular aS accumulation is suppressed not only by d41-50 but also by coexpression of d31-40 and WT, and coexpression of d81-90 and WT. The effects of deletion mutants with shortened d41-50 regions were also examined. Specifically, expression plasmids were constructed for deletion mutants d43-48 (amino acid residues 43-48 deleted), d43-46 (amino acid residues 43-46 deleted), and d43-45 (amino acid residues 43-45 deleted) in SEQ ID NO: 1, and cells were prepared in the same manner as above, separated into sup and ppt, and analyzed by immunoblotting. As a result, as shown in Figure 8, coexpression of d43-45 and d43-46 suppressed seed-dependent aS accumulation by approximately 20%, while the effect of d43-48 was approximately 40%.

[0051] Furthermore, we investigated the effect of d41-50 expression on aS accumulation when insolubilized aS aggregates prepared from patient brains were introduced into cells as seeds, rather than recombinant WT seeds. WT and deletion mutants such as d41-50 were transiently expressed in SH-SY5Y cells. Then, insolubilized aS aggregates (MSA seeds) prepared from the brains of multiple system atrophy (MSA) patients were introduced into the cells and cultured for 3 days. The cells were harvested and homogenized in the presence of the detergent sarkosyl. They were separated into sarkosyl-soluble and -insoluble fractions by ultracentrifugation, and each was analyzed by immunoblotting. When MSA seeds were introduced into WT-expressing cells, the plasmid-derived WT was phosphorylated and accumulated intracellularly in a seed-dependent manner (Figure 9, bottom panel, lane 2 of the ppt fraction). The 15 kDa band represents WT, and several bands above it represent ubiquitinated WT and multimers. When the amount of the band in lane 2 in the ppt fraction (aS accumulation) was taken as 100%, the accumulation of phosphorylated WT in the ppt fraction was reduced by approximately 90% in cells transiently co-expressing d41-50 with WT (Fig. 9, bottom panel, lane 6 in the ppt fraction). On the other hand, in cells co-expressing other deletion mutants, the accumulation of WT was suppressed by a maximum of approximately 20%.

[0052] Similarly, we investigated the effect of deletion mutants on aS accumulation when insolubilized aS aggregates prepared from the brains of dementia with Lewy bodies (DLB) patients were introduced into cells as seeds (DLB seeds). WT and deletion mutants such as d41-50 were transiently expressed in SH-SY5Y cells. DLB seeds were then introduced into cells and cultured for 3 days. After harvesting, cells were separated into sarkosyl-soluble and -insoluble fractions, each of which was analyzed by immunoblotting. When DLB seeds were introduced into WT-expressing cells, the plasmid-derived WT was phosphorylated and accumulated intracellularly in a seed-dependent manner (Figure 10, bottom panel, lane 2 of the ppt fraction). The 15 kDa band represents WT, and several bands above it represent ubiquitinated WT and multimers. When the amount of the band in lane 2 in the ppt fraction (amount of aS accumulated) was taken as 100%, it was revealed that in cells transiently co-expressing WT and d41-50, the amount of phosphorylated WT accumulated in the ppt was reduced by approximately 40% (Figure 10, bottom panel, lane 6 of the ppt fraction).

[0053] These results demonstrate that the aS deletion mutant d41-50, in particular, strongly suppresses seed-dependent accumulation of WT in vitro and in cultured cells. This is a novel result, as no such report has been published to date. Currently, development of drugs (disease-modifying drugs) that suppress aggregate formation as treatments for neurodegenerative diseases is progressing. In particular, lecanemab, approved in Japan last year as a disease-modifying drug for Alzheimer's disease, has attracted attention. To date, no disease-modifying drugs targeting aS have been commercially available, and development is lagging. The d41-50 discovered by the inventors in this study efficiently suppresses seed-dependent accumulation of WT in vitro. The development of a therapeutic drug targeting seed-dependent accumulation of aS has not been reported to date. Specifically, it is anticipated that expressing aS deletion mutants in patient brains using expression systems such as adeno-associated viruses will suppress aS aggregate formation and exert therapeutic effects, or that expressing aS deletion mutants in asymptomatic healthy individuals will contribute to the prevention of diseases associated with aS accumulation.

[0054] Method of Example 1 (Preparation of Plasmids Encoding Deletion Mutants of aS) Plasmids encoding deletion mutants of aS were prepared using the expression plasmids pRK172-aS and pcDNA3-aS, into which human WT (wild-type aS) cDNA (SEQ ID NO: 2) had been inserted. Some of the primers used are listed below. Using these primers and the wild-type vector described above as a template, various deletion mutant vectors were prepared using the KOD Plus Mutagenesis Kit (Toyobo). All deletion mutants from d1-10 to d131-140 were prepared, including those for which primers are not listed. Similarly, deletion mutants of d43-45, d43-46, and d43-48 were prepared in the same manner, although primers are not listed. = Primer base sequences = Primer for d21-30 (a mutant in which 21-30 amino acid residues of human aS are deleted) FW: GGAAAGACAAAAGAGGGTGTTCTCTATGTA (SEQ ID NO: 13) RV: CTCAGCAGCAGCCACAACTCCCTCCTTGGC (SEQ ID NO: 14) Primer for d31-40 FW: GGCTCCAAAACCAAGGAGGGAGTGGTGCAT (SEQ ID NO: 15) RV: TGCTGCTTCTGCCACACCCTGTTTGGTTTT (SEQ ID NO: 16) Primer for d41-50 FW: GGTGTGGCAACAGTGGCTGAGAAGACCAAA (SEQ ID NO: 17) RV: TACATAGAGAACACCCTCTTTTGTCTTTCC (SEQ ID NO: 18) Primer for d51-60 FW: GAGCAAGTGACAAATGTTGGAGGAGCAGTG (SEQ ID NO: 19) RV: ATGCACCACTCCCTCCTTGGTTTTGGAGCC (SEQ ID NO: 20) Primers for d61-70 FW: GTGACGGGTGTGACAGCAGTAGCCCAGAAG (SEQ ID NO: 21) RV: TTTGGTCTTCTCAGCCACTGTTGCCACACC (SEQ ID NO: 22) = Nucleotide sequences encoding human aS (WT) and its deletion mutants = Wild type (WT): SEQ ID NO: 2ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGAGTTGTGGCTGCTGAGAAAACCAAACAGGGTGTGGGCAGAAGCAGCAGGAAAGACAAAGAGGGTGTTCTCATGTAGGCTCAAAACCAAAGGAGGGAGTGGTGCATGGTGTGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGGAGGGACGGTGACGGGTGTGGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCTAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGCAGCAGTGGGCAAGAATGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTAGAGGAGGGTATCAAGACTACGAACCTGAAGCCTAA ・D21-31: Sequence number 8 ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGGAGTTGTGGCTGCTGCTGAGGGAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAAGGAGGGAGTGGTGCATGGTGTGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGGAGGAGCAGTGGTGGACGGGTGTGGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCATTGCAGCAGCCCACTGGCTTTGTCAAAAAGCAGCAGTGGGCAAGAATGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTAGAGGAGGGTATCAAGACTACGAACCTGAAGCCTAA ・T31-40: Sequence number 9ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGGAGTTGTGGCTGCTGAGAAAACCAAACAGGGTGTGGGCAGAAGCAGCAGGCTCCAAAACCAAGGAGGAGTGGTGCATGGTGTGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGGAGGGACGGTGACGGGTGTGGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGCAGCAGTGGGCAAGAAGAAGGACCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTAGAGGAAGGGTATCCAAGACTACGAACCTGAAGCCTAA ・D41-50: Sequence number 10 ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGAGTTGTGGCTGCTGCTGGAGAAAACCAAACAGGGTGTGGGCAGAAGCAGCAGGAAAGACAAAGAGGGTGTTCTCTATGTAGGTGTGGCAACAGTGGCTGAGAAGACCAAAGAGCAAGTGACAAATGTTGGGAGGAGCAGTGGTGGACGGGTGTGGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCATTGCAGCAGCCCACTGGCTTTGTCAAAAAGCAGCAGTGTGGCAAGAATGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAGGGTATCAAGACTACGAACCTGAAGCCTAA ・D51-60: Sequence number 11ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGAGCAAGTGACAAATGTTGGAGGAGCAGTGGTGACGGGTGTGACAG CAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA・d61-70: Sequence number 12 ATGGATGTATTCATGAAAGGACTTTCAAAGGCCAAGGAGGGAGTTGTGGCTGCTGCTGAGAAAACCAAACAGGGTGTGGCAGAAGCAGCAGGAAAGACAAAAGAGGGTGTTCTCTATGTAGGCTCCAAAACCAAGGAGGGAGTGGTGCATGGTGTGGCAACAGTGGCTGAGAAGACCAAAGTGACGGGTGTGACAGCAGTAGCCCAGAAGACAGTGGAGGGAGCAGGGAGCATTGCAGCAGCCACTGGCTTTGTCAAAAAGGACCAGTTGGGCAAGAATGAAGAAGGAGCCCCACAGGAAGGAATTCTGGAAGATATGCCTGTGGATCCTGACAATGAGGCTTATGAAATGCCTTCTGAGGAAGGGTATCAAGACTACGAACCTGAAGCCTAA = Amino acid sequences of human aS (WT) and its deletion mutants = Wild type (WT): SEQ ID NO: 1 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA d21-30: SEQ ID NO: 3MDVFMKGLSKAKEGVVAAAEGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA d31-40: SEQ ID NO: 4 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA d41-50: SEQ ID NO: 5 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA d51-60: SEQ ID NO: 6 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA d61-70: SEQ ID NO: 7 MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPPDNEAYEMPSEEGYQDYEPEA

[0055] (Preparation of Recombinant aS Monomer) Expression plasmids encoding human wild-type or mutant aS were transformed into Escherichia coli BL21 / DE3 (Merck, Cat. #69450) and cultured overnight at 37°C on LB plates supplemented with 50 μg / mL sodium ampicillin (Fujifilm Wako Pure Chemical Industries, Cat. #012-23303). The cells were harvested and added to 500 mL of LB medium supplemented with sodium ampicillin to a final concentration of 50 μg / mL. After 4 hours of incubation, isopropyl-β-thiogalactopyranoside (IPTG, Fujifilm Wako Pure Chemical Industries, Cat. #094-05144) was added to a final concentration of 0.6 mM and cultured for an additional 4 hours. The culture was transferred to a centrifuge tube and centrifuged at 2,700 xg for 8 minutes at 4°C. The cells were harvested and stored frozen at -80°C. The cells were then thawed and suspended in 10 mL of aS purification buffer (50 mM Tris-HCl, pH 7.5 / 1 mM EGTA / 1 mM EDTA / 1 mM DTT), transferred to a centrifuge tube, and sonicated on ice for approximately 1 minute. The suspension was centrifuged at 26,600 xg at 4°C for 15 minutes, and the supernatant was collected. 50 μL of 2-mercaptoethanol was added and the mixture was heat-treated at 100°C for 5 minutes. The mixture was returned to ice to cool, and then centrifuged at 26,600 xg at 4°C for 15 minutes. The supernatant was then loaded onto a 2 mL Q Sepharose Fast Flow (Cytiva, Cat. # 17051001) column (column volume: 2 mL) pre-equilibrated with aS purification buffer and washed with 20 mL of aS purification buffer. The column was then washed with 6 mL of aS purification buffer containing 0.1 M NaCl, and then 6 mL of aS purification buffer containing 0.35 M NaCl was added to the column to elute the adsorbed protein. Ammonium sulfate was added to the eluate to 50% saturation, and the column was left on ice for 15 minutes to precipitate the protein. The eluate was centrifuged at 26,600 g for 15 minutes at 4°C, the supernatant was removed, and the resulting precipitate was dissolved in 30 mM Tris-HCl, pH 7.5. The eluate was dialyzed overnight against the same buffer and centrifuged at 163,000 g for 20 minutes at 4°C to remove insoluble material.The solution was then analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC: Agilent Technologies) using a Brownlee Aquapore RP-300 Column (30 mm x 4.6 mm ID, PerkinElmer, Cat. #07110055) to determine the protein concentration, which was designated as recombinant aS monomer.

[0056] (Preparation of aS aggregates (WT aggregates)) 200 μL of purified recombinant aS monomer solution (3-7 mg / mL) was placed in a 1.5 mL tube, and sodium azide was added to a final concentration of 0.1%. This was shaken for 194 min using a shaker (Taitec NR-3) placed in a 37°C incubator. -1 The mixture was incubated for one week with shaking at a rotation speed of 100 rpm. After one week, the monomer solution solidified into a jelly-like substance, which was centrifuged at 163,000 g for 20 minutes to recover the aS aggregates as a precipitate. 200 μL of saline was added to the solution, and the solution was centrifuged in the same manner to remove the remaining monomer. The aS aggregates obtained as a precipitate were resuspended in 200 μL of saline and sonicated (Taitec VP-050 and Branson Sonifier SFX). A 5 μL aliquot was mixed with 15 μL of 6 M guanidine hydrochloride, and the protein concentration of the aS fibrils was measured using RP-HPLC.

[0057] (Seed-dependent aggregation assay of recombinant aS monomer itself) 100 μL of wild-type or deletion mutant aS monomer (1 mg / mL), 10 μL of 1 M Hepes, pH 7.5, 10 μL of 400 μM thioflavin T (ThT, Fujifilm Wako Pure Chemical Industries, Cat. # 202-01002), and 5 μL of wild-type aggregates (WT seed, 0.2 mg / mL) were mixed in a 96-well plate. The fluorescence intensity of ThT was continuously measured at 37°C using an infinite M200 PRO plate reader (TECAN) at an excitation wavelength of 442 nm and an emission wavelength of 485 nm.

[0058] (Inhibition of seed-dependent aggregation of WT monomer by deletion mutant aS) 50 μL of WT monomer (2 mg / mL) was mixed with 50 μL of WT or d41-50 monomer (2 mg / mL) or 50 μL of saline in a 96-well plate. 10 μL of 1 M Hepes, pH 7.5, 10 μL of 400 μM ThT, and 2 μL of WT aggregates (1 mg / mL) were added. The ThT fluorescence intensity of each sample was continuously measured at 37°C using an infinite M200 PRO plate reader (TECAN) at an excitation wavelength of 442 nm and an emission wavelength of 485 nm.

[0059] Human neuroblastoma cells (SH-SY5Y strain, purchased from the American Type Culture Collection, Cat. #CRL-2266) were cultured in Dulbecco's modified eagle's medium nutrient mixture (DMEM) / F-12HAM (Sigma-Aldrich, Cat. #D8062-500ML) supplemented with 10% (v / v) fetal bovine serum, non-essential amino acid solution (MEM Non-Essential Amino Acids Solution (100X), ThermoFisher, Cat. #11140050), and penicillin-streptomycin-glutamine solution (Penicillin-Streptomycin-Glutamine (100X), ThermoFisher, Cat. #10378016) in a 5% CO2 incubator (Thermo Scientific) at 37°C. Collagen-coated 6 cm Petri dishes (BD Biocoat) and 6-well plates (BD Biocoat) were used for culturing. Cell passage was performed when the cells were 100% confluent using the following procedure. After removing the medium from the 6 cm Petri dish, the cells were washed with 1.5 mL of saline, and the saline was removed. 1 mL of 0.25% trypsin was added and the mixture was incubated at 37°C for 5 minutes. 2 mL of fresh medium was then added to stop the trypsin reaction, after which the cells were thoroughly suspended and seeded into a 6 cm Petri dish containing 3 mL of medium. Typically, 4-6 x 10 5 When cells were added to a 6 cm dish containing 3 mL of medium, they became nearly 100% confluent (2-3 × 10 6 (cells / mL).

[0060] (Expression of aS plasmid and introduction of recombinant aS aggregates into cells) The seeding activity of aS aggregates using cultured cells was determined according to the method of Nonaka et al. (Reference 1). 8 x 10 per well in a 6-well plate was used. 5Cells were seeded and cultured overnight. The next day, the WT expression plasmid (pcDNA3-aS) was introduced into the cells using X-treamGENE9 (Roche, Cat. # 6365809001). Specifically, Opti-MEM (ThermoFisher, Cat. # 31985062), plasmid, and X-treamGENE9 were gently mixed at a ratio of 100 μL:1 μg:3 μL and incubated at room temperature for 15 minutes. The mixture was then added dropwise to the culture medium in each well. After 3-5 hours, 1.2 μL of recombinant aS aggregates (0.2 mg / mL) was added to the culture medium in each well. The treated cells were incubated in a CO2 incubator and harvested after several days as described below.

[0061] (Immunoblotting detection of insolubilized aS) The culture medium from each well was removed using an aspirator, and 1 mL of saline was added to the well to detach and recover the cells. Cells were harvested by centrifugation at 1,800 g for 5 minutes. 300 μL of 1% sarkosyl (N-lauroyl sarcosine sodium salt, Sigma-Aldrich, Cat. #L5125-500G) in A68 buffer (10 mM Tris-HCl, pH 7.5 / 1 mM EGTA / 10% sucrose / 0.8 M NaCl) was added, and the cells were disrupted by sonication using a TAITEC VP-050 ultrasonicator (PWM 17% intensity) for 40–60 seconds. Then, 300 μL of 1% sarkosyl in A68 buffer was added, and the cells were centrifuged at 163,000 g for 20 minutes (himac CS100GXL, Eppendorf Himac Technologies). 300 μL of the resulting supernatant (sarkosyl supernatant fraction: sup) was collected and 75 μL of 5x SDS sample buffer (5xSB) containing 5% 2-mercaptoethanol was added. 15 μL of the supernatant was used to quantify the protein content of the supernatant fraction using a BCA assay (BCA Protein Assay Kit, ThermoFisher, Cat. #23225). The precipitate fraction was then sonicated and heated at 100°C for 5 minutes, after which the sarkosyl-insoluble fraction (ppt) was collected.

[0062] The collected samples (sup and ppt) were electrophoresed on a 13.5% polyacrylamide gel and then transferred to a PVDF membrane (Millipore) at 200 mA for 1 hour. The PVDF membrane was blocked in saline containing 3% gelatin (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. #077-03155) for 10 minutes at room temperature, and then incubated with primary antibodies (anti-phosphorylated α-synuclein monoclonal antibody (pSyn#64): anti-64 antibody, 1:1,000 dilution, Fujifilm Wako Pure Chemical Industries, Ltd., Cat. #015-25191) recognizing phosphorylated aS (phosphorylation of Ser at residue 129) and anti-α-synuclein monoclonal antibody (pSyn#64): anti-64 antibody, 1:1,000 dilution, Fujifilm Wako Pure Chemical Industries, Ltd., Cat. #015-25191) recognizing the C-terminus (residues 131-140) of aS, diluted in saline containing 10% bovine serum (CS: Bovine Serum, ThermoFisher, Cat. #16170-078) and 0.1% NaN3 (10% CS / saline). The PVDF membrane was incubated overnight at room temperature with a secondary antibody (1:500 dilution: Biotin-Goat anti-mouse IgG, Vector, Cat. # BA-9200-1.5 or Biotin-Goat anti-rabbit IgG, Vector, Cat. # BA-1000-1.5) diluted in 10% CS / saline. The membrane was then washed with several mL of saline and incubated for 2 hours at room temperature. The membrane was then incubated for 1 hour with peroxidase-labeled avidin-biotin complex (ABC Standard Kit, Vector, Cat. #PK-4000), washed with saline, and incubated with 0.1% 3,3'-Diaminobenzidine (Sigma-Aldrich, Cat. #D8001-5G), 0.2% CS / saline. The protein bands on the membrane were developed by treatment with saline containing 100 mg / mL nickel(II) chloride hexahydrate (Fujifilm Wako Pure Chemical Industries, Ltd., Cat. #141-01045) and 0.05% H2O2 (hydrogen peroxide solution, Sigma-Aldrich Japan, Cat. #13-1910-5). The color development reaction was stopped by washing the PVDF membrane with tap water.(Reference 1) Nonaka T. et al: Seeded aggregation and toxicity of α-synuclein and tau: cellular models of neurodegenerative diseases. J. Biol. Chem. 285(45): 34885-98 (2010).

[0063] Example 2 Overview of Example 2 (Suppression of seed-dependent accumulation by expression of deletion mutant of α-synuclein in wild-type mouse brain) In Example 1, we found that a mutant (d41-50) lacking amino acid residues 41-50 of α-synuclein (aS) suppresses seed-dependent accumulation of wild-type (WT) aS in vitro and in cultured cells. Therefore, we further investigated whether expression of d41-50 also suppresses seed-dependent accumulation of aS in wild-type mouse brain.

[0064] To express the defective mutant aS in mouse brain, an adeno-associated virus (AAV) encoding d41-50 was constructed (AAV-d41-50: 1x10 ９ vg / μL) and inoculated into the striatum of the right brain of wild-type mice (2 μL: 2 × 10 ９ After one month of viral inoculation, the mice were inoculated with recombinant aS fibers (seed: 2.5 mg / mL, 4 μL: 10 μg) into the striatum of the right brain. Three months later, the mouse brains were removed and fixed in formalin. The fixed brains were embedded in paraffin blocks and thin sections were prepared. These sections were immunohistochemically stained with anti-phosphorylated aS antibody (pS129) to observe and quantify the aS accumulated in the brain.

[0065] <Contents and Results of Example 2> From the stained image of mouse brain (Figure 11), in the brain (control) expressing AAV encoding GFP (AAV-GFP), numerous pS129-positive intracellular aS aggregates were observed in the inoculated striatum and cortex. This indicates that the recombinant aS fibers (seed) inoculated into wild-type mouse brains functioned as seeds in the brain and induced the accumulation of endogenous mouse aS. On the other hand, when seed was introduced into wild-type mouse brains that had previously expressed AAV-d41-50, the number of pS129-positive aS aggregates in the inoculated striatum and cortex was significantly reduced.

[0066] The area ratio of pS129-positive staining (phosphorylated aS aggregates) in the brain was calculated and the results are shown in Figure 12. As a result, it was found that the area ratio of pS129-positive staining was lower in the brains of AAV-d41-50-expressing mice than in the brains of AAV-GFP-expressing mice in the injector-contralateral striatum and cortex. These results demonstrate that expression of d41-50 in the brain suppresses seed-dependent aS accumulation in vivo.

[0067] <Method of Example 2> (Cultured Cells) HEK293T cells were used for AAV production. The culture medium was DMEM (Dulbecco's Modified Eagle's Medium (High Glucose), Sigma-Aldrich, #D5796-500ML) supplemented with 10% (v / v) fetal bovine serum, non-essential amino acid solution (MEM Non-Essential Amino Acids Solution (100X), ThermoFisher, Cat. #11140050), penicillin-streptomycin-glutamine solution (Penicillin-Streptomycin-Glutamine (100X), ThermoFisher, Cat. #10378016), and sodium pyruvate (GIBCO, #11360). The cells were cultured under 5% CO ２ The cells were cultured at 37°C in an incubator (Thermo Scientific).

[0068] (Construction of AAV Expression Vector) AAV expression vectors were prepared by PCR using pcDNA3-aS d41-50 (a mutant aS lacking amino acid residues 41-50) as a template, and the mutant aS sequence was introduced into the EGFP portion of AAV-hsyn-EGFP (Addgene, #114213) containing the synapsin promoter. The PCR product was electrophoresed on an agarose gel, and the band of interest was excised and DNA extracted using NucleoSpin Gel and PCR Clean-up (Takara, U0609B). The extracted DNA was circularized using the In-Fusion HD Cloning kit (Takara, #639648) to create the expression vector (AAV-hsyn-d41-50).

[0069] (Preparation of AAV) HEK293T cells were placed in ten 10 cm dishes at a concentration of 2.0 × 10 ６The cells were seeded at 1000 x g for 1 hour. A mixture of AAV vector (AAV-hsyn-GFP or AAV-hsyn-d41-50), AAV-PHP.eB vector (a plasmid that determines the AAV serotype), AAV helper plasmid pAdDeltaF6 (Addgene, #112867), and PEI (Polysciences, Inc.) was added to the cells. Specifically, for each 10 cm dish, 2.5 μg of AAV vector, 5 μg of pΔF6, 2.5 μg of AAV-PHP.eB, and 50 μL of 1 mg / mL PEI were added to 1 mL of Opti-Mem (Gibco Opti-MEM #11058-021) and mixed, followed by incubation at room temperature for 20 minutes. The entire mixture was then added to the culture medium in the dish for transfection. After two days, the entire medium was replaced with serum-free DMEM medium. Four days later, the medium was collected and centrifuged at 4,500 rpm for 5 minutes to remove the cells. The medium was then filtered through a 0.45 μm filter (Thermo Nalgene Rapid-Flow Filters, #165-0045) to remove excess cell debris, transferred to a centrifuge tube (Millipore Amicon Ultra, #UFC910024), and centrifuged at 5,000 g until approximately 800 μL remained on the membrane. Next, 10 mL of PBS was added to the membrane, and the tube was centrifuged at 5,000 g until approximately 800 μL remained on the membrane. This procedure was repeated three times, and the virus solution on the membrane was collected and stored frozen at -80°C.

[0070] (AAV Titer Measurement) The WPRE sequence, an AAV-specific sequence, was amplified by real-time PCR using the following primers: FW: SEQ ID NO: 23 TGGCGTGGTGTGCACTGT RV: SEQ ID NO: 24 AGGGACGTAGCAGAAGGACG For the PCR sample, 2 μL of the AAV sample was mixed with PCR alkaline treatment buffer (25 mM NaOH, 0.2 mM EDTA) and heat-treated at 100°C for 10 minutes using a thermal cycler (BioRAD). 50 μL of PCR neutralization buffer (1 M Tris-HCl, pH 5.0) was then added and mixed by pipetting. 2 μL of this mixture was mixed with 10 μL of SYBR Green (Thermo Scientific, #4367659), 2 μL of primers, and 6 μL of DW, and analyzed by real-time PCR. The ABI 7500 Fast (Thermo Fisher, #4406984) was used as the instrument, and the conditions were 25°C for the first 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds.

[0071] The plasmid concentration and copy number were calculated using the following formula: [Plasmid concentration ng / μL (1.2 × 10^15)] / (Number of bases bp * 607.4) + 157.9] AAV-hsyn-EGFP (Addgene #114213), whose plasmid concentration and number of bases were known, was serially diluted to prepare a standard curve of copy number versus reaction time, and the copy number (vector genome: vg) was calculated from the reaction time of the purified virus solution.

[0072] (AAV Infection of Mice) Male wild-type mice (C57BL / 6J) were purchased from Japan SLC Co., Ltd. and bred in the animal breeding facility of the Tokyo Metropolitan Institute of Medical Science.

[0073] The samples inoculated into the mouse brain were as follows: 1. AAV-hsyn-EGFP, 1.0 x 10 ９ vg / μL to 2 μL (2.0 x 10 ９ vg: negative control) 2. AAV hsyn-d41-50, 1.0 × 10 ９ vg / μL to 2 μL (2.0 x 10 ９vg) Mice were placed in an anesthesia chamber, filled with anesthetic (20% isoflurane, Japanese Pharmacopoeia, Pfizer) at 3%, and left to stand for several minutes. The anesthetized mice were then secured with auxiliary ear bars and further administered isoflurane inhalation anesthesia at 2%. The four AAV samples were inoculated into the striatum of both mice as follows: First, the mouse's scalp was incised to locate the bregma, and a hole was drilled 2 mm to the left and right and 0.5 mm vertically from that position. The needle of a syringe (HAMILTON, Cat. #80301) containing the sample was inserted to a depth of 3 mm, taking care not to bend it, and 5 μL was inoculated. To reduce sample leakage, the needle was left to stand for 1 minute, then removed, and the opposite hole was inoculated in the same manner. The mice were then sutured, and finally, ear holes were punched for individual identification, and the mice were returned to their cages.

[0074] (Extraction of Mouse Brains) One month after inoculation of the sample, the brains were removed. Mice were anesthetized by intraperitoneal injection of 0.5 mL of a triple-component anesthesia solution. The triple-component anesthesia solution was prepared by mixing 750 μL of medetomidine hydrochloride (Domitor, Nippon Zenyaku Kogyo Co., Ltd.), 800 μL of midazolam (Sandoz Co., Ltd.), 1 mL of butorphanol tartrate (Betorfar, Meiji Seika Pharma Co., Ltd.), and 7.45 mL of saline (Japanese Pharmacopoeia, Otsuka Saline Injection, Otsuka Pharmaceutical Factory, Inc.). After anesthesia, the whole body was bled with saline (Terumo Saline, Type B, Terumo), and the brain was removed. The removal was performed carefully using surgical scissors and tweezers to avoid damaging the tissue.

[0075] (Preservation of mouse brains) The excised mouse brains were immediately placed on a petri dish and divided into left and right halves along the midline using a single-edged feather steel blade (black blade). The right brain was frozen on dry ice and stored at -80°C. The left brain was fixed at 4°C in 10% neutral buffered formalin (Fujifilm Wako Pure Chemical Industries, Cat. #062-01661). Tissue fixation was carried out as quickly as possible (for 2-3 days) because not only does the tissue decay over time, but there is also a risk of the target antigen leaking out.

[0076] (Paraffin Block Embedding) After fixing, the mouse brain was placed in a paraffin block embedding cassette (SAKURA Tissue-Tek #4143) and immersed in paraffin using an automated paraffin embedding system (Sakura Seiki Co., Ltd., Tissue-Tek VIP5 Junior) according to the protocol for channel 1. The mouse brain and liquid paraffin were then poured into a metal paraffin embedding mold and solidified at 0°C to produce a paraffin block. (Thin Section Preparation and Immunohistochemical Analysis) The mouse brain embedded in the paraffin block was sliced ​​to a thickness of 8 μm using a thin-sectioning system (Yamato Kogyo Co., Ltd., Ritratome, #REM-710) equipped with a tissue blade holder (Yamato Kogyo Co., Ltd., Hard Tissue Blade Holder, #BH-220) and a microtome blade (Feather, Microtome Blades, #S35 TYPE). They were attached to anti-peeling coated slide glass (MATSUNAMI). Next, the sections were soaked in xylene for 20 minutes, and then soaked in ethanol for 20 minutes to dissolve the paraffin. The sections were washed with running water for 5 minutes to wash away the remaining organic solvent, and then autoclaved in 0.01 M sodium citrate buffer at 121°C for 20 minutes. Then, they were soaked in 3% H ２ O ２The sections were immersed in PBS containing 0.03% Triton X-100 and washed three times with PBS. Blocking was performed for 20 minutes with 10% bovine serum (CS: Bovine Serum, ThermoFisher, Cat. #16170-078) containing 0.03% Triton X-100, followed by overnight incubation with a 1:1,000 diluted phospho-aS specific antibody (anti-pS129, Abcam, Cat. #ab51253). The next day, the sections were washed three times with PBS and incubated for 2 hours at room temperature with a secondary antibody (1:1,000 diluted: Biotin-Goat anti-mouse IgG, Vector, Cat. #BA-9200-1.5 or Biotin-Goat anti-rabbit IgG, Vector, Cat. #BA-1000-1.5). Afterwards, the sections were washed three times with PBS and reacted with avidin-biotin-peroxidase complex using ABC Kit (peroxidase-labeled avidin-biotin complex (ABC Standard Kit, Vector, Cat. #PK-4000) at room temperature for 30 minutes. After washing three times with PBS, the sections were incubated with the prepared color developing solution (0.1% 3,3'-Diaminobenzidine (Sigma-Aldrich, Cat. #D8001-5G) and 0.05% H ２ O ２ The sections were then stained for 20 minutes in Tris-saline (containing hydrogen peroxide solution, Sigma-Aldrich Japan, Cat. #13-1910-5). The color reaction was stopped by rinsing the slides with tap water. After drying, the sections were treated with Mayer's Hematoxylin (Muto Chemicals, Cat. #30002) for 1 minute to stain the nuclei, followed by 5 minutes of rinsing in running water. The specimens were completely dried, immersed in xylene for 10 minutes, dehydrated, and mounted in Antifade Mounting Medium for Fluorescence (VECTASHIELD, Cat. #H-1000-10). The prepared slides were stored at room temperature. An all-in-one microscope (KEYENCE: BZ-X710) was used for observation, and the area of ​​the anti-pS129-positive structures (phosphorylated aS aggregates) that appeared in the mouse brain was calculated, and the proportion of these was taken as the amount of aggregates.

[0077] The present invention relates to a technology for inhibiting the accumulation of α-synuclein, and is extremely useful, for example, in research and medical applications related to the accumulation of α-synuclein.

Claims

1. A composition for inhibiting alpha-synuclein accumulation, comprising a deletion mutant of alpha-synuclein or a nucleic acid construct that expresses the deletion mutant.

2. The composition of claim 1, wherein the deletion mutant of alpha-synuclein is missing one or more but not more than 40 consecutive amino acids.

3. The composition of claim 2, wherein the deletion mutant of alpha-synuclein is missing 3 or more but not more than 15 consecutive amino acids.

4. The composition of claim 1, which inhibits seed-dependent alpha-synuclein accumulation.

5. The composition described in claim 2, wherein the deletion of consecutive amino acids is a deletion in the region corresponding to the 21st to 70th amino acids or the 81st to 120th amino acids in the amino acid sequence of alpha-synuclein shown in Sequence No.

1.

6. The composition described in claim 5, wherein the deletion of consecutive amino acids is a deletion in the region corresponding to amino acids 35 to 55 in the amino acid sequence of alpha-synuclein shown in SEQ ID NO:

1.

7. The composition of claim 1, for use in treating or preventing alpha-synucleinopathy.

8. A deletion mutant of alpha-synuclein which is missing a sequence of 3 or more but not more than 30 consecutive amino acids, the deletion of the consecutive amino acids being in the region corresponding to the 21st to 70th amino acids or the region corresponding to the 81st to 120th amino acids in the amino acid sequence of alpha-synuclein shown in SEQ ID NO: 1, and which is missing at least the amino acids corresponding to the 43rd and / or 44th amino acids in the amino acid sequence shown in SEQ ID NO: 1, or at least the amino acids corresponding to the 103rd and / or 104th amino acids.

9. A nucleic acid construct encoding the deletion mutant of claim 8.

10. A method for suppressing seed-dependent accumulation of alpha-synuclein, the method comprising the step of coexisting a deletion mutant of alpha-synuclein with wild-type alpha-synuclein.

11. A method for screening a substance that inhibits seed-dependent alpha-synuclein accumulation, comprising a step of detecting whether aggregation of wild-type alpha-synuclein is inhibited in the coexistence of wild-type alpha-synuclein, a candidate substance, and alpha-synuclein seeds.

Images