Agent for suppressing propagation and / or aggregation of modified protein, therapeutic and / or prophylactic agent for neurodegenerative disease, and method for screening for said therapeutic and / or prophylactic agent

Abstract

Provided are: a protein that is modified by a non-hereditary or hereditary neurodegenerative disease; an agent for suppressing propagation and / or aggregation of a protein that is modified by a hereditary neurodegenerative disease; a therapeutic and / or prophylactic agent for neurodegenerative disease, the therapeutic and / or prophylactic agent containing the aforementioned agent; and a method for screening for the aforementioned therapeutic and / or prophylactic agent. The present invention relates to: an agent for suppressing propagation and / or aggregation of a modified protein containing at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or the UBL3 protein, and substances that enhance the activity of the UBL3 protein; and a therapeutic and / or prophylactic agent for a modified protein aggregation disease, the therapeutic and / or prophylactic agent containing the aforementioned agent.

Description

Agent for inhibiting propagation and / or aggregation of denatured proteins, agent for treating and / or preventing neurodegenerative diseases, and method for screening said agent for treating and / or preventing diseases

[0001] The present invention relates to an agent for inhibiting the propagation and / or aggregation of proteins that denature in non-genetic or genetic neurodegenerative diseases (preferably denatured protein aggregation diseases), a therapeutic and / or preventive agent for neurodegenerative diseases containing said agent, and a method for screening said therapeutic and / or preventive agent.

[0002] The propagation and / or aggregation of denatured proteins is known to cause various neurodegenerative diseases. There is a social need to improve the propagation and / or aggregation of denatured proteins and to treat and / or prevent diseases caused by the propagation and / or aggregation of denatured proteins. Furthermore, an agent for treating and / or preventing diseases caused by the propagation and / or aggregation of denatured proteins can maintain a high quality of life for patients after treatment, and is a subject of great social need, including their rehabilitation into society. As a technique for inhibiting the propagation and / or aggregation of denatured proteins, for example, in the case of prion proteins, there is a method of reducing normal prion proteins, but this has the drawback of impairing the function of normal prion proteins.

[0003] On the other hand, exosomes are small extracellular vesicles (hereinafter simply referred to as "sEVs") derived from multivesicular bodies (hereinafter simply referred to as "MVBs"). The intercellular delivery of proteins by exosome-containing sEVs is known to be associated with neurodegenerative diseases, etc. Proteins are generally known to undergo various post-translational modifications by various post-translational modification factors, and such post-translational modifications can affect various cellular processes. Ubiquitin-like protein 3 (hereinafter simply referred to as "UBL3") is known to contain a ubiquitin-like (UBL) domain, is evolutionarily conserved in animals, filamentous fungi, and plants, and is known to be a membrane protein localized by prenylation. The present inventors have found that post-translational modification by UBL3 can sort proteins into sEVs (e.g., Patent Document 1).

[0004] Patent No. 7284447

[0005] Chen, B. , Hasan, M. M. , Zhang, H. ,Zhai,Q. , Waliullah, A. S. M. , Ping, Y. 、. .. .. &Setou, M. (2023). UBL3 Interacts with Alpha-synuclein in Cells and the Interaction is Downregulated by the EGFR Pathway Inhibitor Osimertinib. Biomedicines, 11(6), 1685.

[0006] Furthermore, the present inventors have found that UBL3 interacts with α-synuclein, and have found that UBL3 may be a new therapeutic target for synucleopathy (hereinafter also referred to simply as "synucleopathy") (Non-Patent Document 1). However, at the time of publication of Non-Patent Document 1, it was unclear whether UBL3 contributed to the onset of synucleopathy or to the suppression of synucleopathy, and the present inventors rather speculated that UBL3 contributed to the onset of synucleopathy (i.e., was the cause of synucleopathy).

[0007] In view of the above-mentioned problems of the conventional art, an object of the present invention is to provide an agent for inhibiting the propagation and / or aggregation of proteins (such as synuclein, 43 kDa transactivation response DNA-binding protein (TAR DNA-binding protein 43: TDP43), tau, etc.) that denature in non-genetic or genetic neurodegenerative diseases (preferably denatured protein aggregation diseases) and proteins (such as huntingtin protein, etc.) that denature in genetic neurodegenerative diseases (such as hereditary Parkinson's disease and Huntington's chorea), an agent for treating and / or preventing neurodegenerative diseases containing the agent, and a method for screening for the agent for treating and / or preventing the agent.

[0008] As a result of extensive research into the above-mentioned problems, the present inventors focused on UBL3 as a new target for diseases caused by denatured proteins and found that substances that enhance UBL3 suppress the aggregation and / or propagation of denatured proteins. Furthermore, they found that activation of the UBL3 extracellular vesicle system can selectively alleviate the aggregation and / or propagation of denatured proteins, enabling treatment without reducing normal proteins. Based on these findings, they found that substances that enhance UBL3 have therapeutic and / or preventive effects (e.g., pathological) on neurodegenerative diseases and other diseases caused by denatured proteins. The present invention has been completed based on the above-mentioned findings. Specifically, the present invention is as follows.

[0009] <1> An agent for inhibiting the propagation and / or aggregation of denatured proteins, comprising at least one selected from the group consisting of a substance that enhances the expression of the UBL3 gene or the UBL3 protein, and a substance that enhances the activity of the UBL3 protein. <2> The agent according to <1>, wherein the substance that enhances the expression of the UBL3 gene or the UBL3 protein comprises a vector containing the UBL3 gene. <3> The agent according to <1>, wherein the substance that enhances the activity of the UBL3 protein comprises a substance that enhances the interaction between the UBL3 protein and a denatured protein. <4> The agent according to <1>, wherein the denatured protein is denatured α-synuclein, TDP43, tau, huntingtin protein, or amyloid beta. <5> An agent for treating and / or preventing a denatured protein aggregation disease, comprising the agent according to <1> above. <6> The agent according to <5>, wherein the denatured protein aggregation disease is at least one selected from the group consisting of synucleopathy, TDP43 dyscrasia, tauopathy, Huntington's disease, and Alzheimer's disease. <7> A pharmaceutical composition comprising the agent according to <1> or <5> above.

[0010] <8> A screening method for a therapeutic and / or prophylactic agent for a denatured protein aggregation disease, the method comprising a step of screening using enhanced expression of the UBL3 gene or UBL3 protein, or enhanced activity of the UBL3 protein, as an index. <9> The method according to <8>, wherein the activity of the UBL3 protein is an activity of the UBL3 protein interacting with a denatured protein. <10> The method according to <8>, wherein the denatured protein aggregation disease is at least one selected from the group consisting of synucleopathy, TDP43opathy, tauopathy, Huntington's disease, and Alzheimer's disease.

[0011] <11> A method for inhibiting the propagation and / or aggregation of denatured proteins, comprising at least one selected from the group consisting of enhancing the expression of the UBL3 gene or UBL3 protein and enhancing the activity of the UBL3 protein. <12> A method for treating and / or preventing denatured protein aggregation disorders, comprising at least one selected from the group consisting of enhancing the expression of the UBL3 gene or UBL3 protein and enhancing the activity of the UBL3 protein.

[0012] According to the present invention, it is possible to provide an agent for inhibiting the propagation and / or aggregation of proteins (synuclein, TDP43, tau, etc.) that degenerate in non-genetic or genetic neurodegenerative diseases (preferably denatured protein aggregation diseases) and proteins (e.g., huntingtin protein, etc.) that degenerate in genetic neurodegenerative diseases (e.g., hereditary Parkinson's disease, Huntington's chorea, etc.), a therapeutic and / or preventive agent for neurodegenerative diseases containing the agent, and a method for screening the therapeutic and / or preventive agent.

[0013] Figure 1 shows fluorescence microscopy images of misaggregated synuclein induced by thioflavins. Bar = 50 μm. Figure 2 shows immunohistochemical staining images of α-synuclein (phosphorylated S129) in wild-type (WT) mice and Ubl3-KO mice (C57BL6J) after PFF administration. Figure 3 shows immunohistochemical staining images of α-synuclein (phosphorylated S129) in wild-type (WT) mice and Ubl3-KO mice (C57BL6J) after PFF administration. Figure 4 shows immunohistochemical staining images of α-synuclein (phosphorylated S129) in A53T mice after adeno-associated viral vector (AAV) treatment. Figure 5 shows immunohistochemical staining images of α-synuclein (phosphorylated S129) in A53T mice after adeno-associated viral vector (AAV) treatment. (A) Immunohistochemical staining images of α-synuclein (phosphorylated S129) in Ubl3 knockout / Tg(Thy1-SNCA) and Ubl3 knockout / Tg(Prnp-SNCAA53T) mice and wild-type Tg(Thy1-SNCA) and Tg(Prnp-SNCAA53T) mice. (B) Schematic diagram of split-Gluc-tagged proteins including the N-terminal fragment of huntingtin protein containing an expanded polyglutamine (HTTpolyQ78) and NGluc-UBL3, NGluc-UBL3Δ5, and nHTTpolyQ78-CGluc. (B) (C) Luminescence of culture medium and cell lysate from transfected HEK293 cells. Luminescence ± standard deviation (S.D.) from three independent experiments. Figures showing the interaction of UBL3 with TDP43. (A) Schematic diagram of TDP43 protein and split Gluc-tagged proteins, including NGluc-UBL3 and NGluc-UBL3CAAX-del. (B) Luminescence from transfected HEK293 cells. Figures showing significant reductions in UBL3 expression in synucleopathy patients compared to healthy controls. Figures showing an overview of screening using LIGHTHOUSE. Figures showing drug screening results at 100 nM using a cell-free system split Gluc assay (NGluc-UBL3 + SNCA-CGluc).Figure 1 shows the results of Western blot analysis of α-synuclein (crude extract, TBS soluble fraction, SDS soluble fraction, urea soluble fraction). Figure 2 shows the results of Western blot analysis of α-synuclein (crude extract, TBS soluble fraction, SDS soluble fraction, urea soluble fraction). Figure 3 shows the results of Western blot analysis of α-synuclein (crude extract, TBS soluble fraction, SDS soluble fraction, urea soluble fraction). Figure 4 shows the results of Western blot analysis of α-synuclein (crude extract, TBS soluble fraction, SDS soluble fraction, urea soluble fraction). Figure 5 shows the results of Western blot analysis of α-synuclein (crude extract, TBS soluble fraction, SDS soluble fraction, urea soluble fraction). Figure 6 shows the results of quantification of tyrosine hydroxylase-positive neurons in the substantia nigra of Ubl3-KO, Ubl3-KO / SNCA, and Ubl3-KO / A53T mice. Figure 1 shows the results of live cell imaging of BV2 cells transiently transfected with an mStayGold-Ubl3 expression vector. Figure 2 shows the results of immunocytochemical staining (scale bar: 25 μm). Figure 3 shows the results of live cell imaging of mStayGold-Ubl3 expression in BV2 cells after 30 μM CQ treatment (scale bar: 50 μm). Figure 4 shows the results of live cell imaging of SRAI-Ubl3 expression in BV2 cells after 30 μM CQ treatment. Figure 5 shows the results of live cell imaging of mStayGold-Ubl3 expression in BV2 cells treated with 30 μM CQ and supplemented with 100 nM Lysotracker red. Figure 6 shows the results of live cell imaging of mStayGold-Ubl3 expression in BV2 cells treated with 30 μM CQ and supplemented with 100 nM Lysotracker red. 1 is a diagram showing the results of degradation of aggregates by UBL3-AAV in an α-synuclein-injected model mouse. 2 is a diagram showing the results of degradation of aggregates by UBL3-AAV in an α-synuclein-injected model mouse. 3 is a diagram showing the results of degradation of aggregates by UBL3-AAV in an α-synuclein-injected model mouse. 4 is a diagram showing the results of UBL3-AAV degradation of aggregates in a cultured cell system in which α-synuclein has been aggregated. 5 is a diagram showing immunohistochemical staining images of HD patients and a control group. 6 is a diagram showing the results of analysis of the interaction between UBL3 and mHTT. 7 is a diagram showing the results of analysis of the interaction between UBL3 and mHTT.1 shows the results of quantifying intracellular and extracellular mHTT by HiBiT assay, and 2 shows the results of localization analysis by immunocytochemical staining.

[0014] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.

[0015] In this specification and claims, "denatured protein" refers to an abnormal protein that has denatured and become structurally abnormal. Examples of denatured proteins include misfolded (incorrectly folded) abnormal proteins, abnormal proteins that cannot fold into their normal structure, and thermally denatured proteins. More specifically, examples include abnormal proteins that have lost their intended function and abnormal proteins that have acquired toxic functions. In this specification and claims, "aggregation" refers to the phenomenon in which a large number of denatured proteins gather together and the resulting structure. Aggregation of misfolded abnormal proteins is preferred. It is also preferred that "aggregation" refers to loss of the protein's intended function. Proteins form a folded three-dimensional structure specific to their amino acid sequence. However, not all proteins can easily fold into a three-dimensional structure; sometimes, multiple partially folded proteins become entangled, forming a structure known as an aggregate. This aggregation not only results in the loss of the protein's function, but is also known to be involved in diseases (e.g., denatured protein aggregation disease).

[0016] In this specification and claims, "propagation" refers to the amplification within a cell of a seed denatured protein (preferably a misfolded abnormal protein, more preferably an aggregated abnormal protein) (e.g., by converting a protein with a normal structure into the same abnormal protein as itself, etc.), and the movement, propagation, and / or transmission between cells (preferably between neurons, more preferably a neural circuit), such as prion-like propagation. Propagation may be by diffusion, but examples include transsynaptic transport between neurons, and propagation via a neural circuit is preferred.

[0017] In this specification and claims, the term "denatured protein aggregation disease" refers to a disease caused by a denatured protein (preferably aggregation of a denatured protein), and examples thereof include proteopathies (synucleopathy, TDP43opathy, tauopathy, etc.).

[0018] In this specification and claims, "degeneration" in neurodegenerative disease means that nervous tissue or nerve cells change or regress from an organized state in which they are engaged in normal activity to a lower state (e.g., an abnormal state that deviates from normal organization), and neurodegenerative disease refers to a group of diseases that exhibit various degenerative changes centered on nerve cells.

[0019] The UBL3 gene or UBL3 protein and a method for obtaining the same are described below. (UBL3 Protein) In the present specification and claims, the UBL3 protein refers to any of the proteins described in (a) to (c) below: (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2 in the Sequence Listing; (b) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 in the Sequence Listing, in which one or more amino acids have been deleted, substituted, and / or added, and which has the activity of modifying (e.g., post-translationally modifying) any protein (including the activity of interacting with a denatured protein), the activity of sorting (transporting or distributing) any protein to MVB or sEV by the modification, the activity of excreting any protein extracellularly by the modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing as a membrane protein (e.g., by prenylation, preferably by prenylation of CAAX, more preferably by prenylation of CVIL); (c) A protein consisting of an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 or 2 in the Sequence Listing, and having the activity of modifying an arbitrary protein (including the activity of interacting with a denatured protein), the activity of sorting an arbitrary protein into MVB or sEV by said modification, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing the arbitrary protein as a membrane protein. Here, C in CAAX represents cysteine, A represents a hydrophobic amino acid, and X represents an arbitrary amino acid. Proteins derived from human or mouse can be used as is, and no additional transformation or the like is required. The protein (a) above is preferred. The activity of modifying an arbitrary protein specifically refers to the activity of binding to an arbitrary protein via at least one amino acid residue at positions 110 to 117 of SEQ ID NO: 1 or 2 (preferably positions 111 to 117, more preferably positions 111 to 116, even more preferably positions 112 to 115, and particularly preferably positions 113 or 114). SEQ ID NO: 1 represents the amino acid sequence of human UBL3 protein.SEQ ID NO: 2 represents the amino acid sequence of the mouse UBL3 protein.

[0020] As used herein, the term "one to several" in the context of "an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added" is not particularly limited, but preferably refers to about 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. As used herein, "an amino acid sequence having 90% or more homology" means that the amino acid homology is 90% or more, and the homology is preferably 93% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 98% or more. Proteins encoded by mutant genes highly homologous to the gene having the nucleotide sequence set forth in SEQ ID NO: 3 or 4 in the Sequence Listing, and which have the activity of modifying any protein (including the activity of interacting with a UBL3 protein and a denatured protein), the activity of sorting any protein into MVB or sEV by said modification, the activity of excreting any protein extracellularly by said modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing as a membrane protein, are all within the scope of the present invention. Although the side chains of amino acids that constitute proteins differ from one another in terms of hydrophobicity, charge, size, etc., several highly conserved relationships that do not substantially affect the three-dimensional structure (also called steric structure) of the entire protein have been known empirically and through physicochemical measurements. For example, substitutions of amino acid residues include glycine (Gly) and proline (Pro), Gly and alanine (Ala) or valine (Val), leucine (Leu) and isoleucine (Ile), glutamic acid (Glu) and glutamine (Gln), aspartic acid (Asp) and asparagine (Asn), cysteine ​​(Cys) and threonine (Thr), Thr and serine (Ser) or Ala, lysine (Lys) and arginine (Arg), etc.

[0021] Therefore, even if a mutant protein is one resulting from a substitution, insertion, deletion, or the like in the amino acid sequence of UBL3 set forth in SEQ ID NO: 1 or 2 in the Sequence Listing, as long as the mutation is highly conserved in the three-dimensional structure of UBL3 and the mutant protein has the activity of modifying an arbitrary protein in a manner similar to UBL3 (including the activity of interacting between the UBL3 protein and a denatured protein), the activity of sorting an arbitrary protein into MVB or sEV by said modification, the activity of excreting an arbitrary protein extracellularly by said modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing as a membrane protein, all of these fall within the scope of UBL3. There are no particular limitations on the method for obtaining the UBL3 protein, and it may be a protein synthesized by chemical synthesis, a naturally occurring protein isolated from a biological sample or cultured cells, or a recombinant protein produced by genetic engineering.

[0022] (UBL3 Gene) In this specification and claims, all genes encoding UBL3 proteins (e.g., proteins having the amino acid sequence represented by SEQ ID NO: 1 or 2) belong to the UBL3 gene. SEQ ID NO: 3 shows the nucleotide sequence of the coding region (CDS) encoding the human UBL3 gene. SEQ ID NO: 4 shows the CDS nucleotide sequence encoding the mouse (Mus musculus (house mouse)) UBL3 gene.

[0023] Specific examples of the UBL3 gene include genes described in either (d) or (e) below, and from the viewpoint that a human- or mouse-derived gene can be used as is and no additional transformation or the like is required, the gene described in (d) below is preferred: (d) a gene consisting of the nucleotide sequence described in SEQ ID NO: 3 or 4 in the Sequence Listing, (e) a gene encoding a protein consisting of the nucleotide sequence described in SEQ ID NO: 3 or 4 in the Sequence Listing in which one or several nucleotides have been deleted, substituted, and / or added, and which has the activity of modifying an arbitrary protein (including the activity of interacting with a denatured protein), the activity of sorting an arbitrary protein into MVB or sEV by the modification, the activity of excreting an arbitrary protein extracellularly by the modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing as a membrane protein

[0024] As used herein, the range of "one or several" in the "base sequence in which one or several bases are deleted, substituted and / or added" is not particularly limited, but preferably means about 1 to 20, more preferably 1 to 10, and even more preferably 1 to 5. The degree of DNA mutation mentioned above includes, for example, DNA having 80% or more homology with the base sequence of the UBL3 gene set forth in SEQ ID NO: 3 or 4 in the Sequence Listing, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 98% or more homology. As described above, even if the DNA sequence of the UBL3 gene set forth in SEQ ID NO: 3 or 4 in the Sequence Listing is partially altered by various artificial processes, such as site-directed mutagenesis, random mutation by treatment with a mutagen, or mutation, deletion, or ligation of DNA fragments by restriction enzyme cleavage, these DNA variants are within the scope of the UBL3 gene, regardless of their difference from the DNA sequence set forth in SEQ ID NO: 3 or 4, as long as they encode a protein having the activity of modifying an arbitrary protein, the activity of sorting an arbitrary protein into MVB or sEV by the above-mentioned modification, the activity of excreting an arbitrary protein extracellularly by the above-mentioned modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing the protein as a membrane protein.

[0025] (Obtaining the UBL3 gene) There are no particular limitations on the method for obtaining the UBL3 gene. The UBL3 gene can be isolated by preparing appropriate probes and primers based on the information on the amino acid and nucleotide sequences set forth in SEQ ID NOS: 1 to 4 in the sequence listing herein, and using them to select a desired clone from a human cDNA library (prepared by standard methods from appropriate cells in which the UBL3 gene is expressed). The UBL3 gene can also be obtained by PCR.

[0026] A gene (mutant gene) encoding a protein consisting of the base sequence set forth in SEQ ID NO: 3 or 4 in the Sequence Listing described above in this specification, in which one or several bases have been deleted, substituted, and / or added, and which has the activity of modifying an arbitrary protein (including the activity of interacting with a UBL3 protein and a denatured protein), the activity of sorting an arbitrary protein into MVB or sEV by the above modification, the activity of excreting an arbitrary protein extracellularly by the above modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing the protein as a membrane protein, can also be prepared by any method known to those skilled in the art, such as chemical synthesis, genetic engineering techniques, or mutagenesis.

[0027] <Agent for inhibiting the propagation and / or aggregation of denatured proteins> A first aspect of the present invention is an agent for inhibiting the propagation and / or aggregation of denatured proteins, comprising at least one substance selected from the group consisting of a substance that enhances the expression of the UBL3 gene or the UBL3 protein, and a substance that enhances the activity of the UBL3 protein.

[0028] In this specification and claims, "enhancement" means enhancement of the expression of the UBL3 gene or UBL3 protein, and / or enhancement of the binding of the UBL3 protein to a denatured protein, and / or enhancement of the excretion (preferably extracellularly) of the denatured protein by the UBL3 protein, and / or enhancement of the degradation of the denatured protein by the UBL3 protein. The present inventors have invented a therapeutic and / or prophylactic agent that inhibits the aggregation and / or propagation of denatured proteins and alleviates symptoms in individuals by enhancing the expression of the UBL3 gene or UBL3 protein or enhancing the activity of the UBL3 protein. When cells were administered with, for example, preformed fibrils of synuclein (PFF), this therapeutic and / or prophylactic agent exhibited the effect of suppressing aggregation of denatured proteins in the UBL3-strongly expressing cell line, whereas aggregation of denatured synuclein proteins was amplified in the parent cell line and the UBL3-KO cell line (see Example 1, Figure 1). This indicates that overexpression of UBL3 suppresses aggregation of denatured proteins.

[0029] Furthermore, administration of PFF to the mouse brain exacerbated the propagation and aggregation of denatured proteins in Ubl3-KO mice compared to WT mice (see Example 2, Figure 2), suggesting that UBL3 has a function to inhibit propagation and / or aggregation.

[0030] Furthermore, we demonstrated that enhancing UBL3 using an adeno-associated virus reduced denatured proteins, such as denatured α-synuclein, in the mouse brain (see Example 3, Figure 3). This was effective in TgSNCA[A53T] mice, which develop hereditary Parkinson's disease, suggesting that this method may also be effective in preventing the propagation of synuclein aggregation in hereditary diseases.

[0031] Furthermore, we have shown that UBL3 binds to the huntingtin protein, which aggregates in the genetic disease Huntington's disease, and secretes it extracellularly (see Example 5, Figure 5), and that UBL3 also binds to TDP43, the protein responsible for amyotrophic lateral sclerosis, and secretes it extracellularly (see Example 6, Figure 6).

[0032] In the present invention, examples of denatured proteins include denatured α-synuclein (an example of denatured α-synuclein is synuclein PFF), TDP43, tau (an example of denatured tau is tau aggregates), huntingtin protein, amyloid β, and the like. Examples of synuclein PFF include fibers (or aggregates or assemblies) consisting of a large number of misfolded α-Syn protein repeats (particularly, 25 to several hundred misfolded α-Syn protein repeats). Examples of tau aggregates include molecular complexes containing two or more tau monomers.

[0033] In the present invention, the at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or the UBL3 protein and substances that enhance the activity of the UBL3 protein includes a vector containing the UBL3 gene, a compound having a nitrogen-containing heterocyclic structure, a urea bond (carbamide) structure, and / or an amide bond, having 3 to 100 carbon atoms (preferably 4 to 80 carbon atoms, more preferably 5 to 60 carbon atoms, and even more preferably 6 to 50 carbon atoms), a nucleic acid molecule (preferably an aptamer), an antibody (a polyclonal antibody, a monoclonal antibody), etc. The vector containing the UBL3 gene is preferably a viral vector, and more preferably an adeno-associated viral vector (AAV). In the compound having 3 to 100 carbon atoms and having a nitrogen-containing heterocyclic structure, a urea bond (carbamide) structure, and / or an amide bond, examples of the nitrogen-containing heterocyclic structure include a pyridine ring structure, a piperidine ring structure, a piperazine ring structure, a phenylpiperazine ring structure, a pyrazole ring structure, a macrolactam structure, and / or a macrolide structure. Examples of the compound having a nitrogen-containing heterocyclic structure, a urea bond (carbamide) structure, and / or an amide bond and having 3 to 100 carbon atoms include any EGFR inhibitor (e.g., osimertinib), any anti-inflammatory agent (e.g., sulfasalazine), any anticancer agent (e.g., gemcitabine), any folate metabolic antagonist (e.g., pemetrexed), any immunosuppressant (e.g., tacrolimus, tacrolimus oxide), any DPP-4 (Dipepeptidyl peptidase-4) inhibitor (e.g., teneligliptin, teneligliptin bromine oxide), any oral multikinase inhibitor (e.g., regorafenib, regorafenib oxide), any atypical antipsychotic (e.g., aripiprazole), and the like.

[0034] More specific preferred examples of substances that enhance the activity of UBL3 protein include tacrolimus, teneligliptin, regorafenib, aripiprazole, and the like.

[0035] Furthermore, an aptamer refers to a nucleic acid molecule composed of single-stranded RNA or DNA, which, due to its three-dimensional structure, can bind to a target protein and act as an agonist (enhancing activity) or antagonist (inhibiting activity) (Drug Delivery System 31-1, 2016, pp. 10-14), and in the present invention, an agonist (enhancing activity) is preferred. Aptamers have high binding affinity and specificity for target proteins, low immunogenicity, can be produced by chemical synthesis (preferably obtained by the SELEX method), and have high storage stability. The SELEX method refers to a method of screening for nucleic acid aptamers that selectively (preferably specifically) bind to a target protein from a mixture of nucleic acids called a nucleic acid library. The base length of an aptamer that selectively binds to UBL3 protein is not particularly limited as long as it specifically binds to UBL3 protein, but is preferably 15 to 60 bases, more preferably 20 to 50 bases, even more preferably 25 to 47 bases, and particularly preferably 26 to 45 bases. Polyclonal antibodies can be prepared by isolating and purifying serum obtained from an animal immunized with an antigen (UBL3 protein). Monoclonal antibodies can be prepared by fusing antibody-producing cells obtained from an animal immunized with the antigen (UBL3 protein) with myeloma cells to produce hybridomas, culturing the hybridomas, or administering the hybridomas to an animal to cause ascites tumorigenesis in the animal, and isolating and purifying the culture medium or ascites fluid.

[0036] The degree of enhancement in the enhancing substance compared to the control condition includes an enhancement of 1.2 times or more, a statistically significant enhancement, etc., with an enhancement of 1.5 times or more being more preferable, an enhancement of 2 times or more being even more preferable, an enhancement of 3 times or more being particularly preferable, and an enhancement of 4 times or more being most preferable. There is no particular upper limit to the degree of enhancement, but examples include 30 times or less, 20 times or less, 10 times or less, etc.

[0037] Here, "control conditions" include conditions in the absence of the enhancing substance (for example, a system (e.g., a wild-type system) before administration of the enhancing substance, or a negative control system (a control system administered with a substance that does not affect the expression or activity of the UBL3 gene or protein)), and refer to the use of the difference in response intensity (the "at least one selected from the group consisting of enhanced expression of UBL3 protein or gene (including enhanced cell number of UBL3-expressing cells, UBL3-expressing knockout cells, or wild-type cells), and enhanced activity of UBL3 protein") depending on the presence or absence of the enhancing substance as an indicator. The response intensity S2 under the control conditions may be a value measured before addition of the enhancing substance, or a statistical value or range obtained by collecting data in advance.

[0038] Furthermore, the strength of the response caused by the UBL3 dominant-negative strongly expressing AAV used in each example described below may or may not be used as a "control condition" as a benchmark.

[0039] Furthermore, the activity of inhibiting the propagation and / or aggregation of denatured proteins may be measured by analyzing the reduction of denatured proteins in tissues and / or cells by any conventional method such as immunohistochemical techniques (e.g., staining).

[0040] In this case, the degree of reduction may be 3 / 4 or less, statistically significant inhibition, etc., compared to the control condition, preferably 1 / 2 or less, more preferably 1 / 4 or less, even more preferably 1 / 10 or less, and it is particularly preferable that the denatured protein is eliminated.

[0041] The UBL3 of the present invention includes those derived from mammals such as mice, rats, hamsters, guinea pigs, dogs, pigs, monkeys, and primates, including humans. Preferably, it is human UBL3.

[0042] <Therapeutic and / or preventive agent for denatured protein aggregation disorders> A second aspect of the present invention is a therapeutic and / or preventive agent for denatured protein aggregation disorders, comprising the agent according to the first aspect. The therapeutic and / or preventive effect of the agent according to the second aspect for denatured protein aggregation disorders may be a clinical therapeutic and / or preventive effect or a pathological therapeutic and / or preventive effect. The therapeutic and / or preventive effect for denatured protein aggregation disorders can be referred to as the activity of inhibiting the spread and / or aggregation of denatured proteins. The activity of inhibiting the spread and / or aggregation of denatured proteins can be measured by analyzing the reduction of denatured proteins in tissues and / or cells by any conventional method, such as immunohistochemical techniques (e.g., staining). In this case, the degree of the reduction, compared to the control conditions described above, can be ¾ or less, a statistically significant reduction, etc., preferably ½ or less, more preferably ¼ or less, and even more preferably 1 / 10 or less, and it is particularly preferred that the denatured protein is completely eliminated. The effective amount of the agent according to the second aspect can be said to be an amount that inhibits the propagation and / or aggregation of denatured proteins, and is preferably an amount at which the above-mentioned degree of reduction in denatured proteins in tissues and / or cells is measured. Specific and preferred examples of the agent according to the second aspect include those similar to those described above in the first aspect. Examples of denatured proteins include those similar to those described above in the first aspect. In the present invention, the denatured protein aggregation disease is at least one selected from the group consisting of synucleopathy (e.g., Parkinson's disease, DLB, MSA, etc.), TDP43 opathy, tauopathy, Huntington's disease, and Alzheimer's dementia.

[0043] <Pharmaceutical composition comprising the agent according to the first aspect and the second aspect> A third aspect of the present invention is a pharmaceutical composition comprising the agent according to the first aspect and the second aspect. The pharmaceutical composition according to the third aspect is capable of treating and / or preventing denatured protein aggregation disorders. Specific and preferred examples of the agent according to the first aspect include those similar to those described above in relation to the first aspect.

[0044] The agents according to the first and second aspects can be formulated with various pharmaceutical additives such as excipients, binders, disintegrants, disintegration inhibitors, anti-caking / adhesion agents, lubricants, absorption / adsorption carriers, solvents, bulking agents, isotonic agents, solubilizers, emulsifiers, suspending agents, thickeners, coating agents, absorption promoters, gelation / coagulation promoters, light stabilizers, preservatives, moisture-proofing agents, emulsifying / suspension / dispersion stabilizers, color inhibitors, oxygen scavengers / antioxidants, flavoring / odor masking agents, colorants, foaming agents, antifoaming agents, soothing agents, antistatic agents, and buffer / pH adjusters to form pharmaceutical compositions (pharmaceutical preparations) according to the third aspect, such as oral preparations (tablets, capsules, powders, granules, fine granules, pills, suspensions, emulsions, liquids, syrups, etc.), injections, eye drops, etc. The various agents described above are formulated by conventional methods.

[0045] Oral solid preparations such as tablets, powders, and granules may contain, for example, excipients such as lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, crystalline cellulose, anhydrous dibasic calcium phosphate, partially pregelatinized starch, corn starch, and alginic acid; binders such as simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethylcellulose, shellac, methylcellulose, ethylcellulose, sodium alginate, gum arabic, hydroxypropylmethylcellulose, hydroxypropylcellulose, water, and ethanol; disintegrants such as dry starch, alginic acid, agar-agar, starch, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose, calcium carboxymethylcellulose, and sodium starch glycolate; The solid preparations may be prepared according to a conventional method using pharmaceutical additives for forming solid preparations, such as disintegration inhibitors such as ethanol, stearic acid, cocoa butter, and hydrogenated oils; anti-caking / anti-adhesion agents such as aluminum silicate, calcium hydrogen phosphate, magnesium oxide, talc, and anhydrous silicic acid; lubricants such as carnauba wax, light anhydrous silicic acid, aluminum silicate, magnesium silicate, hydrogenated oils, hydrogenated vegetable oil derivatives, sesame oil, white beeswax, titanium oxide, dried aluminum hydroxide gel, stearic acid, calcium stearate, magnesium stearate, talc, calcium hydrogen phosphate, sodium lauryl sulfate, and polyethylene glycol; absorption promoters such as quaternary ammonium salts, sodium lauryl sulfate, urea, and enzymes; and absorption / adsorption carriers such as starch, lactose, kaolin, bentonite, anhydrous silicic acid, hydrous silicon dioxide, magnesium aluminometasilicate, and colloidal silicic acid. Furthermore, tablets can be coated with conventional coatings as needed, such as sugar-coated tablets, gelatin-coated tablets, gastric-soluble coated tablets, enteric-coated tablets, and water-soluble film-coated tablets. Capsules are prepared by mixing the various pharmaceuticals listed above and filling them into hard gelatin capsules, soft capsules, etc.Furthermore, aqueous or oily suspensions, solutions, syrups and elixirs can also be prepared in accordance with conventional methods using the various liquid formulation additives described above, such as solvents, bulking agents, isotonicity agents, solubilizing agents, emulsifiers, suspending agents and thickeners.

[0046] Injections may be prepared according to standard methods using pharmaceutical additives for liquid formulations, such as diluents such as water, ethyl alcohol, macrogol, propylene glycol, citric acid, acetic acid, phosphoric acid, lactic acid, sodium lactate, sulfuric acid, and sodium hydroxide; pH adjusters and buffers such as sodium citrate, sodium acetate, and sodium phosphate; stabilizers such as sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, and thiolactic acid; isotonicity agents such as table salt, glucose, mannitol, and glycerin; solubilizers such as sodium carboxymethylcellulose, propylene glycol, sodium benzoate, benzyl benzoate, urethane, ethanolamine, and glycerin; soothing agents such as calcium gluconate, chlorobutanol, glucose, and benzyl alcohol; and local anesthetics.

[0047] Eye drops may be prepared according to a conventional method by appropriately blending, for example, preservatives such as chlorobutanol, sodium dehydroacetate, benzalkonium chloride, cetylpyridium chloride, phenethyl alcohol, methyl parahydroxybenzoate, and benzethonium chloride; buffers such as borax, boric acid, and potassium dihydrogen phosphate; thickeners such as methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, sodium carboxymethylcellulose, and chondroitin sulfate; solubilizers such as polysorbate 80 and polyoxyethylene hydrogenated castor oil 60; stabilizers such as sodium edetate and sodium bisulfite; and isotonic agents such as sodium chloride, potassium chloride, and glycerin.

[0048] The subjects to which the agents according to the first and second aspects and the pharmaceutical composition according to the third aspect are administered include mammals such as humans, cows, horses, dogs, cats, pigs, sheep, rats, and mice, and are preferably humans. The method of administration of the agents according to the first and second aspects and the pharmaceutical composition according to the third aspect is not particularly limited, and is determined appropriately depending on the form of the formulation, the age, sex, and other conditions of the subject (e.g., patient), and the severity of the symptoms of the subject (e.g., patient). The mode of administration of the agents according to the first and second aspects and the pharmaceutical composition according to the third aspect is not particularly limited, and may be administered to the subject (e.g., administration of the above-mentioned effective amount) by, for example, oral administration, injection (intracerebral injection, intramuscular injection, intravenous injection, transdermal injection, intraperitoneal injection, etc.), nasal administration, inhalation, etc. Examples of intracerebral injections include stereotactic intracerebral injections into specific regions of the brain (e.g., the striatum, cerebellum, prefrontal cortex, other cerebral cortices, hippocampus, thalamus, hypothalamus, pineal gland, pituitary gland, midbrain, hindbrain, choroid plexus, etc.), intracerebral transplantation, intraventricular injection, and intrathecal injection. Such administration routes can be appropriately selected by those skilled in the art. The dosage, administration frequency, administration period, etc. can also be appropriately determined by those skilled in the art based on the type, sex, age, symptoms, etc. of the subject. The dosage of the active ingredient (which refers to, for example, a group or ingredient having pharmacological activity in the body of an animal including a human, or a group or ingredient that, when contacted with another substance such as a microbial contaminant, causes a physical or chemical change in the other substance or the active ingredient itself; further, physical or chemical changes referred to here include binding, transfer, rearrangement, addition, elimination, decomposition, cleavage, oxidation, reduction, labeling, color development, luminescence, etc.) of the agent according to the first aspect and the second aspect, and the pharmaceutical composition according to the third aspect is selected appropriately depending on the dosage, the age, sex, and type of disease of the subject (e.g., a patient) and other conditions, and is usually 0.1 to 1000 mg administered once or several times in divided doses per day to a subject (e.g., an adult), and preferably 40 to 500 mg administered once or several times in divided doses per day. The dosage of a vector containing the UBL3 gene is selected appropriately depending on the age, sex, type of disease, and other conditions of the recipient (e.g., patient). Generally, the dosage is 1×10 for a recipient (e.g., adult) with a viral (infectious) titer of 1×10 per day. ７~1 x 10 １４ The transduction unit (TU) can be administered once or in divided doses, preferably 1 x 10 per day. ８ ~1 x 10 １３ TU may be administered once or in divided doses. In addition, typically, 1 × 10 ８ ~1 x 10 １５ GC (gene copy) can be administered once or in divided doses, preferably 1 x 10 per day. ９ ~1 x 10 １２ GC may be administered once or in divided doses.

[0049] <Method for inhibiting propagation and / or aggregation of denatured proteins, and method for treating and / or preventing denatured protein aggregation disorders> The present invention also relates to a method for inhibiting propagation and / or aggregation of denatured proteins, comprising at least one selected from the group consisting of enhancing the expression of the UBL3 gene or UBL3 protein, and enhancing the activity of the UBL3 protein. The present invention also relates to a method for treating and / or preventing denatured protein aggregation disorders, comprising at least one selected from the group consisting of enhancing the expression of the UBL3 gene or UBL3 protein, and enhancing the activity of the UBL3 protein. Examples of denatured proteins include those similar to those described above in the first aspect. Examples of denatured protein aggregation disorders include those similar to those described above in the second aspect.

[0050] The degree of enhancement is preferably 1.2-fold or more enhancement, statistically significant enhancement, etc., compared to the control condition, more preferably 1.5-fold or more enhancement, even more preferably 2-fold or more enhancement, particularly preferably 3-fold or more enhancement, and most preferably 4-fold or more enhancement. There is no particular upper limit to the degree of enhancement, but examples include 30-fold or less, 20-fold or less, and 10-fold or less. Here, "control conditions" include conditions in the absence of the enhancing substance (e.g., a system before administration of the enhancing substance (e.g., a wild-type system), or a negative control (a control system administered with a substance that does not affect the expression or activity of the UBL3 gene or protein)). Suppression of the propagation and / or aggregation of denatured protein may be measured by analyzing the reduction of denatured protein in tissues and / or cells using any conventional method, such as immunohistochemical techniques (e.g., staining). The degree of reduction may be 3 / 4 or less, statistically significant inhibition, etc., compared to the control condition, preferably 1 / 2 or less, more preferably 1 / 4 or less, even more preferably 1 / 10 or less, and it is particularly preferable that the denatured protein is eliminated.

[0051] In the method for inhibiting the propagation and / or aggregation of denatured proteins, the at least one selected from the group consisting of enhancing the expression of the UBL3 gene or the UBL3 protein and enhancing the activity of the UBL3 protein includes contacting or administering (e.g., administering an effective amount of) at least one selected from the group consisting of a substance that enhances the expression of the UBL3 gene or the UBL3 protein and a substance that enhances the activity of the UBL3 protein to a subject in vitro or in vivo. The subject to be contacted may be any cell (e.g., cultured cell). The cells may be any animal (e.g., human, mouse) cells (human breast cancer cells (MDA-MB-231-luc-D3H2LN cells), human embryonic kidney cells (e.g., HEK293 cells, HEK293T cells), human cervical cancer cells (e.g., HeLa cells, HeLa-S3 cells), hamster ovary cells (e.g., CHO cells, CHO-K1 cells), etc.) (e.g., cerebral cortex-derived cells, cerebellum-derived cells, hippocampus-derived cells), or any genetically modified cells. In a method for treating and / or preventing a denatured protein aggregation disorder, the at least one selected from the group consisting of enhancing the expression of the UBL3 gene or the UBL3 protein and enhancing the activity of the UBL3 protein includes administering (e.g., administering an effective amount of) to a subject in vivo at least one substance selected from the group consisting of a substance that enhances the expression of the UBL3 gene or the UBL3 protein and a substance that enhances the activity of the UBL3 protein. The effective amount of the substance can be said to be an amount that inhibits the propagation and / or aggregation of the denatured protein, and is preferably an amount at which the above-mentioned degree of reduction in denatured protein in tissues and / or cells is measured.Specific and preferred examples of at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or the UBL3 protein, and substances that enhance the activity of the UBL3 protein include those similar to those described above in the first aspect, such as a vector containing the UBL3 gene, a compound having a nitrogen-containing heterocyclic structure, a urea bond (carbamide) structure, and / or an amide bond, having 3 to 100 carbon atoms (preferably 4 to 80 carbon atoms, more preferably 5 to 60 carbon atoms, and even more preferably 6 to 50 carbon atoms), a nucleic acid molecule (preferably an aptamer), an antibody (a polyclonal antibody, a monoclonal antibody), etc. In the above method, the subject and mode of administration are similar to those described above for the agent according to the first and second aspects and the pharmaceutical composition according to the third aspect. For example, it may be administered to a recipient (including mammals such as humans, cattle, horses, dogs, cats, pigs, sheep, rats, and mice, preferably humans) by oral administration, injection (intracerebral injection, intramuscular injection, intravenous injection, transdermal injection, intraperitoneal injection, etc.), nasal administration, inhalation, or other routes. The dosage is selected appropriately depending on the method of use, the age, sex, type of disease, and other conditions of the recipient (e.g., patient), but typically 0.1 to 1000 mg may be administered to a recipient (e.g., adult) once or several times in divided doses per day, and preferably 40 to 500 mg may be administered once or several times in divided doses per day. Furthermore, when administering a vector containing the UBL3 gene, the dosage is selected appropriately depending on the age, sex, type of disease, and other conditions of the recipient (e.g., patient), but typically 1 x 10 viral (infectious) titer per day for a recipient (e.g., adult). ７ ~1 x 10 １４ The transduction unit (TU) can be administered once or in divided doses, preferably 1 x 10 per day. ８ ~1 x 10 １３ TU may be administered once or in divided doses. In addition, typically, 1 × 10 ８ ~1 x 10 １５ GC (gene copy) can be administered once or in divided doses, preferably 1 x 10 per day. ９~1 x 10 １２ GC may be administered once or in divided doses.

[0052] <Method for screening for therapeutic and / or prophylactic agents for denatured protein aggregation disorders> A fourth aspect of the present invention is a method for screening for therapeutic and / or prophylactic agents for denatured protein aggregation disorders, comprising a screening step using enhanced expression of the UBL3 gene or UBL3 protein (including an increase in the number of UBL3-expressing cells, UBL3-expressing knockout cells, or wild-type cells), or enhanced activity of the UBL3 protein, as an index. As used herein, "screening" means at least narrowing down the population of test substances.

[0053] Examples of denatured proteins include those similar to those described above in the first aspect. Examples of denatured protein aggregation diseases include those similar to those described above in the second aspect. The screening method may be any screening method, such as in vivo, in vitro, or in silico, as long as it uses the above as an indicator. The screening method preferably includes a step of using UBL3-expressing cells, UBL3-expressing knockout cells (UBL3 expression-deficient cells), wild-type cells, a UBL3 gene, or a UBL3 protein. Furthermore, UBL3-expressing cells and wild-type cells may be cells of any animal (e.g., human, mouse) (e.g., cerebral cortex-derived cells, cerebellum-derived cells, hippocampus-derived cells), or may be cells that express UBL3 through genetic engineering, such as human breast cancer cells (MDA-MB-231-luc-D3H2LN cells), human embryonic kidney cells (e.g., HEK293 cells, HEK293T cells), human cervical cancer cells (e.g., HeLa cells, HeLa-S3 cells), and hamster ovary cells (e.g., CHO cells, CHO-K1 cells) that have been made to express UBL3 through genetic engineering. Furthermore, UBL3 expression knockout cells (UBL3 expression-deficient cells) may be cells of any animal (e.g., human, mouse) (e.g., cerebral cortex-derived cells, cerebellum-derived cells, hippocampus-derived cells), or may be cells in which UBL3 expression has been knocked out by genetic engineering, such as human breast cancer cells, human fetal kidney cells, human cervical cancer cells, or hamster ovary cells in which UBL3 expression has been knocked out by genetic engineering.

[0054] Examples of the activity of the UBL3 protein include the activity of modifying any protein (including the activity of interacting between the UBL3 protein and a denatured protein), the activity of sorting any protein into MVB or sEV by the above-mentioned modification, the activity of excreting any protein extracellularly by the above-mentioned modification, the activity of degrading a denatured protein, the activity of inhibiting the propagation and / or aggregation of a denatured protein, and / or the activity of localizing the protein as a membrane protein, and the like, and the activity of interacting between the UBL3 protein and a denatured protein and / or the activity of inhibiting the propagation and / or aggregation of a denatured protein is preferred.

[0055] The screening method involves culturing UBL3-expressing cells, UBL3-expressing knockout cells, or wild-type cells in the presence and absence of a test substance, and if at least one of the following is detected in the presence of the test substance relative to the absence of the test substance: enhanced expression of the UBL3 protein or gene (including an increased cell number of UBL3-expressing cells, UBL3-expressing knockout cells, or wild-type cells) and enhanced activity of the UBL3 protein, a therapeutic and / or preventive agent for denatured protein aggregation disorders can be screened. The degree of enhancement, compared to control conditions, can be 1.2-fold or greater, or a statistically significant enhancement, with 1.5-fold or greater being more preferred, 2-fold or greater being even more preferred, 3-fold or greater being particularly preferred, and 4-fold or greater being most preferred. There is no particular upper limit to the degree of enhancement, but examples include 30-fold or less, 20-fold or less, and 10-fold or less.

[0056] Here, "control conditions" include conditions in the absence of the test substance (for example, a system (e.g., a wild-type system) before administration of the test substance, or a negative control system (a control system administered with a substance that does not affect the expression or activity of the UBL3 gene or protein)), and refer to the difference in response intensity (the "at least one selected from the group consisting of increased expression of UBL3 protein or gene (including increased cell number of UBL3-expressing cells, UBL3-expressing knockout cells, or wild-type cells), and increased activity of UBL3 protein") depending on the presence or absence of the test substance as an index. The response intensity S2 under the control conditions may be a value measured before addition of the test substance, or a statistical value or range obtained by collecting data in advance.

[0057] For example, when "at least one selected from the group consisting of increased expression of the UBL3 protein or gene (including increased cell number of UBL3-expressing cells, UBL3-expressing knockout cells, or wild-type cells), and increased activity of the UBL3 protein" is observed, the test substance is preferably identified as a candidate therapeutic and / or preventive agent for denatured protein aggregation diseases. For example, the identification is preferably performed using the response intensity S1 in the presence of the test substance as an index, and more preferably by comparing the response intensity S1 with the response intensity S2 under control conditions.

[0058] Furthermore, the strength of the response caused by the UBL3 dominant-negative strongly expressing AAV used in each example described below may or may not be used as a "control condition" as a benchmark.

[0059] Furthermore, the activity of inhibiting the propagation and / or aggregation of denatured proteins may be measured by analyzing the reduction of denatured proteins in tissues and / or cells using any conventional method, such as immunohistochemical techniques (e.g., staining).

[0060] In this case, the degree of reduction may be 3 / 4 or less, a statistically significant reduction, etc., compared to the control condition, preferably 1 / 2 or less, more preferably 1 / 4 or less, even more preferably 1 / 10 or less, and it is particularly preferable that the denatured protein is eliminated.

[0061] Measurement of the expression level of UBL3 protein at the mRNA level can be performed by standard methods such as Northern blot, Southern blot, or RT-PCR. Specifically, standard methods known to those skilled in the art, such as those described in Molecular Cloning, Second Edition, or Current Protocols in Molecular Biology, can be used. Furthermore, measurement of the expression level of UBL3 protein can be performed by standard immunoassays, such as Western blot using an antibody or ELISA. Specifically, standard methods known to those skilled in the art, such as those described in Molecular Cloning, Second Edition, or Current Protocols in Molecular Biology, can be used. Furthermore, based on the nucleotide sequence information of the UBL3 gene, UBL3 gene expression in various human tissues can be detected in silico. Furthermore, UBL3 gene expression in various human tissues can be detected in vivo or in vitro, for example, by using probes or primers containing a partial or complete nucleotide sequence of the gene. The expression of the UBL3 gene can be detected by standard methods such as RT-PCR, Northern blotting, and Southern blotting. The screening method may include a step of performing in silico screening by evaluating the binding between a test substance and the UBL3 protein (and denatured protein) by calculating an arbitrary score using a protein-compound binding model. Examples of the score include the Confidence score (CS) and the Interaction score (IS). Examples of the protein-compound binding model include LIGHTHOUSE (Shimizu H et al. iScience. 25:105314, 2022).

[0062] Examples of methods for detecting at least one selected from the group consisting of an increase in the number of UBL3-expressing cells depending on the presence or absence of a test substance, an increase in the expression of the UBL3 protein or gene, and an increase in the activity of the UBL3 protein include a method for detecting signal transduction activation induced by the binding of a test substance to the UBL3 protein (and denatured protein).

[0063] The screening method for therapeutic and / or preventive agents for denatured protein aggregation diseases according to the fourth aspect is preferably a method of screening therapeutic and / or preventive agents for denatured protein aggregation diseases by BiFC (Bimolecular Fluorescence Complementation), specifically, more preferably comprising: (1) a step of allowing a fusion protein comprising a UBL3 protein or a functional fragment thereof and one of the split luciferase fragments, and a fusion protein comprising the denatured protein and the other split luciferase fragment, to coexist in the presence and absence of a test substance, wherein the luciferase fragments are split so that the luminescent activity of the luciferase is restored by binding to each other, and (2) a step of screening therapeutic and / or preventive agents for denatured protein aggregation diseases using the restored luminescent activity as an index, depending on the presence or absence of the test substance.

[0064] (Test Substance) The test substance is not particularly limited. The test substance may consist of a single component (i.e., a pure substance) or a combination of two or more components (i.e., a mixture). When the test substance is a mixture, the number of components constituting the mixture and their composition ratios are not particularly limited. The test substance may be a known substance or a novel substance. The test substance may be a natural product or an artificial product. The test substance may be any vector containing the UBL3 gene. The type of test substance is not particularly limited, and may be a nucleic acid molecule (preferably an aptamer), an antibody, an individual low-molecular-weight synthetic compound (e.g., alcohols, ketones, aldehydes, ethers, esters, hydrocarbons, sugars, organic acids, nucleic acids, amino acids, peptides, lipids, and various other organic or inorganic components), a compound present in a natural product extract, or a synthetic peptide. Alternatively, the test substance may be a chemical compound library, a phage display library, or a combinatorial (compound) library prepared using combinatorial chemistry techniques. Construction of a chemical compound library is known to those skilled in the art, and commercially available chemical compound libraries can also be used. The test substance is preferably a small molecule compound (e.g., a chemical compound library), a protein, a (poly)peptide, a nucleic acid molecule (preferably an aptamer), or an antibody (monoclonal or polyclonal antibody).

[0065] Examples of low molecular weight compounds (e.g., compound libraries) as test substances include compounds having a nitrogen-containing heterocyclic structure, a urea bond (carbamide) structure, and / or an amide bond, and having 3 to 100 carbon atoms (preferably 4 to 80 carbon atoms, more preferably 5 to 60 carbon atoms, and even more preferably 6 to 50 carbon atoms). Examples of the nitrogen-containing heterocyclic structure include a pyridine ring structure, a piperidine ring structure, a piperazine ring structure, a phenylpiperazine ring structure, a pyrazole ring structure, a macrolactam structure, and / or a macrolide structure. Examples of the small molecule compound (e.g., compound library) include any EGFR inhibitor (e.g., osimertinib), any anti-inflammatory agent (e.g., sulfasalazine), any anticancer agent (e.g., gemcitabine), any antifolate (e.g., pemetrexed), any immunosuppressant (e.g., tacrolimus, tacrolimus oxide), any DPP-4 (Dipepeptidyl peptidase-4) inhibitor (e.g., teneligliptin, teneligliptin bromine oxide), any oral multikinase inhibitor (e.g., regorafenib, regorafenib oxide), any atypical antipsychotic (e.g., aripiprazole), and the like.

[0066] An aptamer is a nucleic acid molecule composed of single-stranded RNA or DNA that, due to its three-dimensional structure, can bind to a target protein and act as an agonist (enhancing activity) or antagonist (inhibiting activity) (Drug Delivery System 31-1, 2016, pp. 10-14). In the present invention, agonists (enhancing activity) are preferred. Aptamers have high binding affinity and specificity to target proteins, low immunogenicity, can be produced by chemical synthesis (preferably by the SELEX method), and have high storage stability. The base length of the aptamer that selectively binds to the UBL3 protein as a test substance is not particularly limited as long as it specifically binds to the UBL3 protein; however, it is preferably 15 to 60 bases, more preferably 20 to 50 bases, even more preferably 25 to 47 bases, and particularly preferably 26 to 45 bases.

[0067] The present invention will be explained in more detail below by showing examples of the present invention, but the present invention is not limited to these examples and various applications are possible within the scope of the technical idea of ​​the present invention.

[0068] Example 1: Using MM cells (triple-negative breast cancer cell line), a UBL3-knockout (KO) line and a cell line with strong UBL3 expression were created as follows. (Creation of MDA-MB-231 UBL3-KO line) MDA-MB-231 (triple-negative breast cancer cell line) was purchased from ATCC as the MM cells. An sgRNA sequence (CAGAATCGTTAGGAGAAAC; SEQ ID NO: 9) targeting UBL3 was incorporated into pSpCas9(BB)-2A-Puro (PX459 plasmid). MDA-MB-231 (70% confluency) seeded in a 10 cm dish was transfected with 12.5 μg of the above PX459 plasmid using lipofection (Lipofectamine 2000). One day later, 3 μg / ml (final concentration) of puromycin was added to DMEM, and selection was carried out for two days. After selection, culture was continued in normal DMEM, and cloning was carried out. 12 clones were collected and KO strains were screened by UBL3 Western blot.

[0069] 1 x 10 ６ The MM parent strain, the UBL3-KO strain, and the UBL3-strongly expressing strain were seeded onto 24-well plates. After 24 hours, a synuclein overexpression plasmid and preformed synuclein fibrils (PFF) were introduced into the cells using X-tremeGENE® HP (Roche). After a further 48 hours, thioflavin S and 4',6-diamidino-2-phenylindole (DAPI) were added, and the cells were observed with an InCellAnalyzer and then analyzed with ImageJ. The results are shown in Figure 1. Figure 1 shows a fluorescence microscope image of abnormally aggregated synuclein induced by thioflavins (bar = 50 μm). In each figure below, "Merge" indicates a merged or superimposed image.

[0070] As shown in Figure 1, the cell number did not change significantly. When synuclein aggregation was labeled with thioflavin S and compared with the parental line, the UBL3-overexpressing line showed a significant reduction in synuclein aggregation induced by PFF administration. In contrast, the UBL3-KO line showed a clear increase in synuclein aggregation compared with the parent line.

[0071] Example 2: 48-week-old wild-type (WT) mice and Ubl3-KO mice (C57BL6J) were injected with 10 μg of PFF into the right striatum using stereotaxic surgery. One month after injection, the brains were removed by perfusion fixation. The removed brains were formalin-fixed and paraffin-embedded, and 10 μm-thick sections were prepared. Immunohistochemical staining was performed according to a protocol previously published in our laboratory (Non-Patent Document 1). After serial deparaffinization, the sections were incubated in 1x phosphate-buffered saline (PBS, 0.1 mol / L, pH 7.4) containing 3% hydrogen peroxide (FUJIFILM, Wako Pure Chemical Industries, Ltd.) for 20 minutes, washed three times with 1x PBS, and then treated with a solution containing 1% bovine serum albumin (Sigma-Aldrich) in 1x PBS for 1 hour at room temperature (e.g., 23°C). The samples were then incubated with the primary antibody (rabbit anti-α-synuclein (phosphorylated S129) antibody (Abcam, ab51253, dilution 1:1000)) at room temperature for 1 hour. After washing three times with 1x PBS, the sections were treated with the secondary antibody (anti-rabbit IgG antibody (H+L), biotinylated (Vector's Goat, BA-1000-1.5, dilution 1:1000)) for 1 hour. After washing again three times with 1x PBS, the sections were treated with avidin-biotin complex (Vector Laboratories) in 1x PBS for 1 hour at room temperature. The reaction was visualized using 3,3'-Diaminobenzidine (DAB; FUJIFILM, Wako Pure Chemical Industries, Ltd.). Finally, the sections were counterstained with hematoxylin (FUJIFILM, Wako Pure Chemical Industries, Ltd.), dehydrated in graded alcohols (FUJIFILM, Wako Pure Chemical Industries, Ltd.) (80%, 90%, 100%), and cleared with xylene (FUJIFILM, Wako Pure Chemical Industries, Ltd.). Sections were cover-slip mounted with Patho Mount (FUJIFILM, Wako Pure Chemical Industries, Ltd.). Images of immunohistochemical staining were captured using a NanoZoomer 2.0HT system (Hamamatsu Photonics). The results are shown in Figures 2-1 and 2-2. 2-1 and 2-2 show immunohistochemical staining images of α-synuclein (phosphorylated S129) after administration of PFF to wild-type (WT) mice and Ubl3-KO mice (C57BL6J).Here, α-synuclein is a protein consisting of 140 amino acid residues encoded by the SNCA gene. α-synuclein is phosphorylated at S129, and it is known that α-synuclein that accumulates in Lewy bodies is highly phosphorylated at S129.

[0072] As is clear from the images showing the results of immunohistochemical staining shown in Figures 2-1 and 2-2, significantly stronger phosphorylated-synuclein immunopositive aggregates (Lewy body-like and Lewy neurite-like structures) were observed in the somatosensory cortex, amygdala, substantia nigra, striatum, and cortex of Ubl3-KO mice compared to wild-type mice. From the results shown above, it can be said that at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or UBL3 protein and substances that enhance the activity of the UBL3 protein can suppress the propagation and / or aggregation of denatured proteins.

[0073] Example 3 A53T mutant mice (strain number "RRID:IMSR_JAX:006823" manufactured by The Jackson Laboratory) were used as transgenic mice overexpressing human A53T mutant α-synuclein. The A53T mutant mice may represent a mouse model of a degenerative protein aggregation disease (e.g., synucleopathy). Viral infection with a viral titer of 4 x 10 was administered to 18-week-old A53T mutant mice. ９ A transduction unit (TU) AAV (VectorBuilder) strongly expressing UBL3 and an AAV (VectorBuilder) strongly expressing UBL3 dominant-negative were injected into the right striatum by stereotaxic surgery, and 15 days later, the brains were excised by perfusion fixation. Immunohistochemical staining was performed on the excised brains as in Example 2, and images were obtained. The results are shown in Figures 3-1 and 3-2. Here, UBL3 dominant-negative refers to a brain in which the UBL3 function is inactivated by deleting the CVIL of CCVIL at the C-terminus of UBL3. Figures 3-1 and 3-2 show immunohistochemical staining images of α-synuclein (phosphorylated S129) after treatment of A53T mice with adeno-associated viral vectors (AAV).

[0074] As is clear from the images showing the immunohistochemical staining results shown in Figures 3-1 and 3-2, overexpression of UBL3 in A53T mice significantly reduced phosphorylated-synuclein immunopositive aggregates in the thalamus, substantia nigra, and granuloscleral cortex compared to the group overexpressing a UBL3 dominant-negative. The results shown above demonstrate that at least one substance selected from the group consisting of a substance that enhances the expression of the UBL3 gene or UBL3 protein and a substance that enhances the activity of the UBL3 protein can inhibit the propagation and / or aggregation of denatured proteins. In other words, at least one substance selected from the group consisting of a substance that enhances the expression of the UBL3 gene or UBL3 protein and a substance that enhances the activity of the UBL3 protein can inhibit the propagation and / or aggregation of denatured proteins, and therefore can treat and / or prevent denatured protein aggregation disorders (e.g., synucleopathies), and can at least pathologically treat and / or prevent denatured protein aggregation disorders (e.g., synucleopathies).

[0075] Example 4 C57BL strain mice were used. Ubl3 knockout (Ubl3- / -) mice (the same as the Ubl3-KO mice (C57BL6J) in Example 2) were obtained from a previously established laboratory colony (Ageta H, Ageta-Ishihara N, Hitachi K, Karayel O, Onouchi T, Yamaguchi H, Kahyo T, Hatanaka K, Ikegami K, Yoshioka Y, Nakamura K, Kosaka N, Nakatani M, Uezumi A, Ide T, Tsutsumi Y, Sugimura H, Kinoshita M, Ochiya T, Mann M, Setou M, Tsuchida K. UBL3 modification influences protein sorting to small extracellular vesicles. Nat Commun. 2018 Sep 26;9(1):3936. doi: 10.1038 / s41467-018-06197-y. PMID: 30258067; PMCID: PMC6158211.) Wild-type (WT) mice of the C57BL / 6J strain (SLC Corporation, Hamamatsu, Japan) were used as controls. Transgenic C57BL / 6N-Tg(Thy1-SNCA) and C57BL / 6J-Tg(Prnp-SNCAA53T) mice were purchased from The Jackson Laboratory and used to prepare double mutant Ubl3 knockout / Tg(Thy1-SNCA) and Ubl3 knockout / Tg(Prnp-SNCAA53T) mice. Here, Tg(Thy1-SNCA) mice refer to transgenic mice in which the human SNCA gene was overexpressed under the control of the mouse Thy1 promoter. Furthermore, Tg(Prnp-SNCAA53T) mice refer to transgenic mice in which the A53T mutant human SNCA gene was overexpressed under the control of the mouse prion protein (Prnp) promoter.

[0076] All mice were housed and bred under a 12-hour light / dark cycle. The genotypes of the mice were confirmed by polymerase chain reaction (PCR) according to our previous report (Ageta, H., Ageta-Ishihara, N., Hitachi, K., Karayel, O., Onouchi, T., Yamaguchi, H., Kahyo, T., Hatanaka, K., Ikegami, K., Yoshioka, Y., et al. (2018). UBL3 modification influences protein sorting to small extracellular vesicles. Nat Commun 9, 3936.) and the protocol provided by Jackson Laboratory. The results are shown in Figure 4A and B. 4A and 4B are images showing the results of immunohistochemical staining of α-synuclein (phosphorylated S129) in Ubl3 knockout / Tg(Thy1-SNCA) and Ubl3 knockout / Tg(Prnp-SNCAA53T) mice and wild-type Tg(Thy1-SNCA) and Tg(Prnp-SNCAA53T) mice. In the figures, the significance levels are *: p<0.05, **: p<0.01, and ***: p<0.001.

[0077] As is clear from the results shown in Figures 4A and 4B (particularly the results shown in Figure 4B), α-synuclein was significantly increased in Ubl3 knockout mice compared to wild-type (WT) mice. Furthermore, α-synuclein was significantly increased in Ubl3 knockout / SNCA double transgenic mice compared to SNCA transgenic mice. Furthermore, α-synuclein was significantly increased in Ubl3 knockout / SNCAA53T double transgenic mice compared to SNCAA53T transgenic mice. From the results shown above, it can be said that at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or UBL3 protein and substances that enhance the activity of the UBL3 protein can inhibit the propagation and / or aggregation of denatured proteins, and can function as a therapeutic and / or preventive agent for denatured protein aggregation diseases (e.g., synucleopathies).

[0078] Example 5: Test of interaction between UBL3 and N-terminal fragments of huntingtin protein containing expanded polyglutamines Figures 5A to 5C show the results of a test of the interaction between UBL3 and N-terminal fragments of huntingtin protein containing expanded polyglutamines by a "split-luciferase complementation assay." Figure 5A is a schematic diagram of split Gluc-tagged proteins including the N-terminal fragment of huntingtin protein containing expanded polyglutamines (HTTP polyQ78) and NGluc (an N-terminal fragment of Gluc protein consisting of amino acids 1 to 92)-UBL3, NGluc-UBL3Δ5, and nHTTP polyQ78-CGluc (a C-terminal fragment of Gluc protein consisting of amino acids 93 to 168). Gaussia princeps luciferase (Gluc) is a secreted enzyme produced by Gaussia princeps. It is a simple luminescence enzyme that catalyzes a luminescent reaction in the presence of only the enzyme and a substrate (luciferin, such as coelenterazine). As shown in Figure 5A, we used the Gaussia princeps luciferase (Gluc) sequence-tagged UBL3 (NGluc-UBL3) plasmid, as well as the NGluc-UBL3Δ5 plasmid, 3xFlag-UBL3 plasmid, and 3xFlag-UBL3Δ5 plasmid, in which the "CCVIL" amino acid sequence in the C-terminal region of UBL3 has been deleted. To tag the C-terminal region of the polyglutamine (polyQ)-containing N-terminal HTT fragment (nHTTpolyQ78-CGluc) with a Gluc sequence, the coding sequence from codons 1 to 156 containing HTT HD exon 1 and 78 CAG repeats was inserted in frame before the CGluc sequence and integrated into the PCI vector between the XhoI and MluI sites. The 6xMYC-nHTTpolyQ78 plasmid was amplified by PCR using the following primers: Forward: 5'-CCGCTCGAGATGGCGACCCTGGAAAGC-3' (SEQ ID NO: 5) Reverse: 5'-GCCTCTAGATTAACATATTGTCAGACAATGATTCACACGG-3' (SEQ ID NO: 6)

[0079] As shown in Figure 5A, after digestion with XhoI and XbaI, the fragment was inserted into the pcDNA3-6xMYC vector after the 6xMYC sequence. Human embryonic kidney (HEK) 293 cells (RIKEN Cell Bank) were cultured in Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). Cell culture was performed at 37°C and 5% CO. ２ The cells were incubated in a humidified incubator at 4°C. Cell cultures were grown in culture plates until they reached 60-80% confluence and then transfected with cDNA plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific). The transfection reagent was diluted in Opit-MEM low-serum medium (Thermo Fisher Scientific) according to the manufacturer's recommendations. The conditioned media (CM) collected from the cells was centrifuged at 1,200 rpm for 5 minutes to remove cell debris. The cells were lysed in 1% Triton® X-100 (Sigma-Aldrich) and centrifuged at 13,000 rpm for 5 minutes to obtain the supernatant. After adding 17 μg / ml of coelenterazine (Cosmo Bio) diluted in Opti-MEM to the samples, luciferase activity was immediately measured using a microplate reader (BioTek). The luciferase activity of untreated DMEM (10% FBS) was set as background. The results are shown in Figures 5B and 5C.

[0080] As is clear from the results shown in Figures 5B and 5C, luminescence was observed both intracellularly and extracellularly upon binding of UBL3 and UBL3Δ5 to huntingtin. The extracellular luminescence observed in Figure 5B suggests that huntingtin bound to UBL3 and UBL3Δ5 is likely excreted extracellularly via exosomes. From the results shown above, it can be said that at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or UBL3 protein and substances that enhance the activity of the UBL3 protein can inhibit the propagation and / or aggregation of denatured proteins, and can function as a therapeutic and / or preventive agent for denatured protein aggregation diseases (e.g., Huntington's disease).

[0081] Example 6: Test for interaction between UBL3 and TDP43 protein Figures 6A and 6B show the results of a test for interaction between UBL3 and TDP43 protein by a "split-luciferase complementation assay." Figure 6A is a schematic diagram of TDP43 protein and split Gluc-tagged proteins including NGluc-UBL3 and NGluc-UBL3CAAX-del, and Figure 6B shows the luminescence results from transfected HEK293 cells. First, as shown in Figure 6A, a 390X plasmid vector was obtained from the Institute of Physical and Chemical Research, in which a nucleic acid sequence encoding the N-terminal fragment "NGluc" of the 1st to 92nd amino acids of the Gluc protein was inserted into a 4006-base pair pCL84 gene expression vector having a CMV promoter and an ampicillin resistance gene, and a 273X plasmid vector was obtained from the same pCL84 gene expression vector, in which a nucleic acid sequence encoding the C-terminal fragment "CGluc" of the 93rd to 168th amino acids of the Gluc protein was inserted. Next, a UBL3 gene was further inserted 3' from the NGluc gene into the 390X plasmid vector to construct plasmid construct A, and a C-terminally deleted UBL3 gene (UBL3CAAX-del) was further inserted 3' from the NGluc gene into the 390X plasmid vector to construct plasmid construct B. A nucleic acid sequence encoding the TDP43 protein was further inserted 5'-side of CGluc in the 273X plasmid vector to construct plasmid construct C. The above constructs (NGluc constructs A, B, CGluc construct C) were introduced individually or in appropriate combinations into HEK293 cells cultured in a 12-well plate. ２ The cells were cultured under 5% CO₂ for 72 hours. 100 μL of the medium was transferred to a 96-well plate. 100 μL of 17 μg / mL coelenterazine was added to the medium on the 96-well plate. The fluorescence intensity of the culture medium and cell lysate was measured. The results are shown in Figure 6B.

[0082] As is clear from the results shown in Figure 6B, the binding of UBL3 to TDP43 protein was confirmed by luminescence using the same method as in Example 5. From the results shown above, it can be said that at least one substance selected from the group consisting of substances that enhance the expression of the UBL3 gene or UBL3 protein and substances that enhance the activity of the UBL3 protein can inhibit the propagation and / or aggregation of denatured proteins, and can function as a therapeutic and / or preventive agent for denatured protein aggregation diseases (e.g., TDP43 opathies).

[0083] Example 7: Demonstration test of significant reduction in UBL3 expression levels in synucleinopathy patients compared to healthy controls. A Mann-Whitney U test (two-tailed test, significance level α = 0.05) was performed on UBL3 and Small Ubiquitin-related Modifier 4 (SUMO4) as a comparison between healthy controls and Parkinson's disease patients (hereinafter also referred to simply as "PD"). GraphPad Prism 10.4.0 was used for statistical analysis. SUMO4 is a small protein with a structure similar to ubiquitin. It belongs to the group of SUMO proteins (small proteins with a structure similar to ubiquitin) that are involved in various physiological processes, mainly through protein modification within cells. SUMO modification is known to regulate the function, stability, localization, etc. of target proteins. SUMO4 has been implicated in the immune system and inflammatory responses, and several studies have shown its association with immune-related diseases, cancer, and other illnesses. Like other SUMO family members, SUMO4 is known to affect cellular responses by modifying target proteins and regulating their function.

[0084] (Materials and Methods) We used GSE205450 (https: / / ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE205450), a public database in the Gene Expression Omnibus (GEO) operated by the National Center for Biotechnology Information (NCBI), which contains an RNA-seq dataset of postmortem human brains with Parkinson's disease (PD). Among the datasets included in GSE205450 (https: / / www.ncbi.nlm.nih.gov / geo / download / ?acc=GSE205450), data processed by NCBI using Transcripts Per Million (TPM) (GSE205450_norm_counts_TPM_GRCh38.p13_NCBI.tsv.gz) was obtained, and the gene expression levels of SUMO4 in the putamen between healthy and PD groups were obtained. For the correspondence between GeneID and gene name, refer to Human.GRCh38.p13.annot.tsv.gz. Sample GSM6212998 contained mixed information on both the healthy and PD groups and was therefore deemed inappropriate and excluded. Finally, gene expression levels in the putamen were obtained for 27 healthy samples and 28 PD samples. The results are shown in Figure 7.

[0085] As is clear from the results shown in Figure 7, there was no significant difference in the gene expression level of SUMO4 between the healthy group and PD patients (p>0.05). On the other hand, the gene expression level of UBL3 was significantly decreased in PD patients (p<0.001). These results suggest that UBL3 may function as a therapeutic and / or preventive agent for disorders involving denatured protein aggregation.

[0086] Example 8 Screening of therapeutic and / or preventive agents for denatured protein aggregation disorders, and the obtained inhibitors of propagation and / or aggregation of denatured proteins as the therapeutic and / or preventive agents (Method 1) First, the confidence score (CS) and interaction score (IS: binding strength score) of a set of existing approved drugs (10,757 types) for UBL3 protein were calculated using LIGHTHOUSE (protein-compound binding model (Shimizu H et al. iScience. 25: 105314, 2022)). Figure 8 shows an overview of screening using LIGHTHOUSE. In Figure 8, compounds T to Z are arbitrary compounds. As outlined in FIG. 8, LIGHTHOUSE was used to screen (search) compounds that bind to UBL3 protein from a set of existing approved drugs (10,757 types).

[0087] (Result 1) For UBL3, 319 compounds with a CS of 0.7 and 403 compounds with a strong binding affinity (>7.0) were found. Of these, 10 compounds had a CS of >0.7 and an IS of >7.0. Excluding compounds identified multiple times in previous LIGHTHOUSE analyses (i.e., compounds that may bind nonspecifically to various proteins), 157 compounds with a CS of >0.7 were found. Seven compounds (effectively four) had an IS of >7.0. Ten compounds in the search space predicted to bind to UBL3 at the significance level (CS >0.7, IS >7.0), seven of which were specific. Three of these compounds were derivatives, so essentially four compounds were found: Tacrolimus, Teneligliptin, Regorafenib, and Aripiprazole.

[0088] (Method 2) For the above four species, Gaussia princeps luciferase complementation assay was carried out using NGluc-UBL3 and α-syn-CGluc plasmids.

[0089] cDNA transfection and cell culture Human embryonic kidney 293 (HEK-293) cells (Riken Cell Bank) were cultured in phenol red-free Dulbecco's modified Eagle's medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich). Cell culture was performed at 37°C and 5% CO. ２ The cells were maintained in a humidified incubator at 47°C. According to the reagent's instructions, cells were cultured in culture plates until 60-70% confluent, and then transiently transfected with cDNA plasmids using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) diluted in Opti-MEM low-serum medium. Sample preparation for cell-free luciferase assay for drug screening: 48 hours after transfection, the cell culture supernatant (CM) was collected and centrifuged at 1200 rpm for 5 minutes. 100 nM of drug (dissolved in DMSO) was added to the collected culture supernatant and incubated at room temperature for 1 hour. The treated culture supernatants were transferred to white Nunc™ MicroWell™ 96-well, Nunclon Delta-Treated, flat-bottom microplates (Thermo Fisher Scientific). Coelenterazine (17 μg / mL, Cosmo Bio, Kyodo) was added to the drug-treated culture supernatants and diluted with Opti-MEM. Luminescence intensity was immediately measured using a microplate reader (BioTek) with an integration time of 1 second. The luminescence intensity of untreated DMEM (10% FBS) was used as background, and the background luminescence intensity was subtracted from all drug-treated and untreated culture supernatants for correction. The results are shown in Figure 9.

[0090] (Result 2) Figure 9 shows the results of drug screening at 100 nM using a cell-free system split Gluc assay (NGluc-UBL3+SNCA-CGluc). As is clear from the results shown in Figure 9, the fluorescence intensity of the four drugs, Tacrolimus, Teneligliptin, Regorafenib, and Aripiprazole, was enhanced compared to the control group (Mock), demonstrating enhanced binding of UBL3 to α-synuclein. These results suggest that Tacrolimus, Teneligliptin, Regorafenib, and Aripiprazole can function as inhibitors of the propagation and / or aggregation of denatured proteins. Furthermore, it can be said that the four drugs, Tacrolimus, Teneligliptin, Regorafenib, and Aripiprazole, can function as therapeutic and / or prophylactic agents for denatured protein aggregation disorders (at least are therapeutic and / or prophylactic agents for denatured protein aggregation disorders).

[0091] Example 9: Confirmation test of significant increase in expression of phosphorylated α-synuclein in Ubl3-deficient mice (quantification of α-syn by various Western blotting) To investigate the effect of Ubl3 on the expression of various α-syns, soluble and insoluble α-syns were extracted stepwise from mouse brain homogenates and subjected to Western blotting (Rina Bandopadhyay, JOVE; 2016).

[0092] (Materials and Methods) All fractions of brain tissue lysates from all six mouse groups were loaded onto a 14% SDS-PAGE gel. Proteins were then transferred to a polyvinylidene fluoride membrane (Cytiva). For phosphorylated α-syn (pS-129), the membrane was blocked with shaking at room temperature for 1 hour in Tris-buffered saline (TBS-T; 100 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5% [v / v] Tween-20) containing 2.5% bovine serum albumin (BSA, Sigma-Aldrich) and 0.5% [w / v] skim milk (Nacalai Tesque) with Tween-20(+) (Fujifilm Wako Pure Chemical Industries, Ltd.). The membrane was then incubated with primary antibodies (pS-129, Ab Chem Inc.; α-syn and β-actin, Cell Signaling Tech. Inc.) overnight at 4°C with shaking. After washing three times with TBS-T, the membrane was incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour with shaking. Immunoreactive proteins were generated using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and detected with a FUSION FX imaging system (Vilber Lourmat, Collégien, Seine-et-Marne). The results are shown in Figures 10-1 to 10-4. Figures 10-1 to 10-4 show the results of Western blot analysis of α-synuclein (crude extract, TBS-soluble fraction, SDS-soluble fraction, and urea-soluble fraction). In Figures 10-1 to 10-4, all graphs (A to H) show the ratio of phosphorylated or wild-type α-synuclein / β-actin quantified using ImageJ and normalized to the housekeeping gene β-actin.

[0093] As is clear from the results shown in Figures 10-1 to 10-4, Western blot analysis of sequentially extracted α-syn (crude extract, TBS-soluble fraction, SDS-soluble fraction, and urea-soluble fraction) confirmed the effect of Ubl3 on α-syn gene expression. Expression of phosphorylated α-syn (pS-129), normalized to the housekeeping gene β-actin, was upregulated in the brains of Ubl3-KO mice, double mutant Ubl3-KO / SNCA, and Ubl3-KO / A53T mice compared with wild-type strains at 1 year of age. These results demonstrate that the expression level of phosphorylated α-synuclein was significantly increased in Ubl3-deficient mice.

[0094] Example 10: Confirmation test of significant reduction in dopaminergic neurons in the substantia nigra of Ubl3-deficient mice Quantification of tyrosine hydroxylase (TH)-positive neurons: The effect of Ubl3 on α-syn expression and the effect of aggregated α-syn on dopaminergic neurons were further evaluated by immunofluorescence experiments using TH antibodies.

[0095] (Materials and Methods) Immunofluorescence: Sections were deparaffinized and rehydrated. Then, to block endogenous peroxidase activity, 3% H ２ O ２The sections were treated with 105°C for 20 minutes. For antigen retrieval, Tris-EDTA buffer (TE; pH 8.0, Nippon Gene) was used and autoclaved at 105°C for 1 minute. The sections were blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich) at room temperature for 1 hour. They were then incubated with a primary antibody (tyrosine hydroxylase, Sigma-Aldrich) overnight at 4°C. The secondary antibody was diluted in 1% BSA-containing PBS and incubated at room temperature for 1 hour. Nuclear staining was performed using DAPI (Cellstain). The sections were then mounted with mounting medium (VECTASHIELD, Vector). Fluorescent images were captured using a confocal laser scanning microscope (Leica TCS SP8, USA) with a 63x objective. The results are shown in Figure 11. Figure 11 shows the quantification of tyrosine hydroxylase-positive neurons in the substantia nigra of Ubl3-KO, Ubl3-KO / SNCA, and Ubl3-KO / A53T mice. Figure 11(A) shows the quantification of TH-positive neurons using ImageJ software, and an unpaired t-test with Welch's correction was performed. Data are shown as mean (n=5) ± standard error of mean (SEM). Figure 11(B) shows immunofluorescent staining of the substantia nigra of mouse brain using a TH antibody (1:1000, Millipore).

[0096] As shown in Figure 11(A) and (B), quantification of tyrosine hydroxylase-positive neurons in the substantia nigra of all mouse strains reflected the role of Ubl3 in α-syn expression. Brain sections from 1-year-old mice showed that TH-positive neurons were reduced in the substantia nigra of Ubl3-KO, Ubl3-KO / SNCA, and Ubl3-KO / A53T mice compared with wild-type, SNCA, and A53T mice.

[0097] Example 11: Test to confirm that UBL3 is degraded in lysosomes in microglial cells (BV2) Tests to confirm that UBL3 is degraded in lysosomes in BV2 cells were conducted based on the following (1) to (3): (1) No detection of Ubl3 expression in BV2 cells, (2) Localization of Ubl3 in BV2 cells, and (3) Inhibition of lysosomal Ubl3 degradation by chloroquine. Regarding (1) above, to observe the expression and intracellular localization of Ubl3, live cell imaging was performed using a confocal microscope with the mStayGold-Ubl3 expression vector. Next, regarding (2) above, to further evaluate the localization of Ubl3 in BV2 cells and confirm whether Ubl3 is actually not expressed or whether mStayGold is subsequently degraded, immunocytochemical staining was performed on BV2 cells using an anti-UBL3 antibody. Regarding (3) above, inhibition of autophagy suggests that lysosomal protein degradation is impaired. We investigated whether chloroquine (CQ) treatment inhibited lysosomal degradation of Ubl3. The results are shown in Figures 12 to 17.

[0098] (Materials and Methods) ((Cell Culture and Treatment)) BV2 cells (provided by Professor Ueki of Nagoya University) were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1% penicillin-streptomycin mixture (Nacalai Tesque). The cells were maintained at 37°C and 5% CO. ２ The cells were cultured in a humidified incubator for 1 hour at 37°C and 5% CO. The medium was then replaced with Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) until the cells reached 60-80% confluence. The cells were then pretreated with chloroquine (CQ; 30 μM; Wako) as an inhibitor for 3 hours. ２The cells were cultured in a humidified incubator for 1 h at 4°C for 1 h at 4°C. Thereafter, the cells were transiently transfected with the plasmid using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) diluted with Opti-MEM (Thermo Fisher Scientific) according to the manufacturer's recommended method.

[0099] Plasmids: mStayGold (primer sequences: forward: 5'-CGCGGATCCATGGTGTGTCTACAGGCGAGGAG-3' (SEQ ID NO: 7); reverse: 5'-CTAGCTAGCCAGGTGGGGCCTCCAGG-3' (SEQ ID NO: 8)) and SRAI were inserted into the UBL3 (NM_007106) expression pcDNA3 vector, located between the BamHI and NheI sites before the UBL3 sequence, using conventional molecular biology techniques and PCR. For the 3xFlag-UBL3 plasmid, the coding sequence for UBL3 was inserted in frame after the 3xFlag sequence in the pcDNA3.1-3xFlag vector, between the BamHI and EcoRI sites.

[0100] (Live Cell Confocal Microscopy) For live cell imaging experiments, BV2 cells were cultured in 35 mm glass-bottom dishes and observed under a confocal microscope at an excitation wavelength of 488 nm. Live cell confocal images were acquired using a confocal imaging system (Leica TCS SP8, Wetzlar, Germany).

[0101] Immunocytochemistry (ICC) staining: First, the culture medium was removed, and the cells were washed with ice-cold PBS. Then, they were fixed with ice-cold 100% paraformaldehyde (PFA) for 5 minutes and washed three times with ice-cold phosphate-buffered saline (PBS). Blocking was performed for 1 hour using 1% bovine serum albumin (BSA) diluted in PBS. Primary antibody was added and incubated overnight at 4°C. After incubation, the cells were washed three times with PBS for 5 minutes each using a dilution of the primary antibody anti-UBL3 antibody (ABclonal, A4028, 1:500). Secondary antibody (Alexa Flour 633, 1:500) was diluted in PBS containing 1% BSA and incubated at room temperature for 1 hour in the dark. Nuclear staining was performed using 4',6-diamidino-2-phenylindole (DAPI, Dojindo Laboratories). After removing the secondary antibody solution, the cells were washed three times with PBS for 5 minutes each time in the dark. Then, the cells were mounted with VECTASHIELD mounting medium (Vector Laboratories). Confocal imaging was performed using a 63x objective on a confocal laser scanning microscope (Leica TCS SP8, Wetzlar, Germany).

[0102] (Live Cell Imaging) For live cell imaging experiments, BV2 cells were incubated in humidified CO ２ The cells were stained with a 100 nM solution of LysoTracker Red DND-99 (Invitrogen) in DMEM for 30 minutes in an incubator, and then observed under a confocal microscope at an excitation wavelength of 615 nm (Texas Red). Live cell confocal images were acquired using a confocal imaging system (Leica TCS SP8, Wetzlar, Germany).

[0103] The results are shown in Figures 12 to 17. Figure 12 shows the results of live cell imaging of BV2 cells transiently transfected with an mStayGold-Ubl3 expression vector (scale bar: 10 μm) for (1) above. In each figure, BF stands for bright field. Figure 13 shows the results of immunocytochemical staining for (1) above (scale bar: 25 μm). Figure 14 shows the results of live cell imaging of mStayGold-Ubl3 expression in BV2 cells treated with 30 μM CQ (scale bar: 50 μm) for (2) above. BV2 cells were pretreated with 30 μM CQ, transfected with mStayGold-Ubl3, and then incubated for 48 hours. Live cell imaging was performed using a confocal microscope. Figure 15 shows the results of live cell imaging of SRAI-Ubl3 expression in BV2 cells treated with 30 μM CQ (scale bar: 50 μm), as described in (2) above. BV2 cells were pretreated with 30 μM CQ, transfected with SRAI-Ubl3, and then incubated for 48 hours. Figure 16 shows the results of live cell imaging of mStayGold-Ubl3 expression in BV2 cells treated with 30 μM CQ and supplemented with 100 nM Lysotracker Red (scale bar: 50 μm), as described in (3). FIG. 17 shows the results of live cell imaging of mStayGold-Ubl3 expression in BV2 cells treated with 30 μM CQ and supplemented with 100 nM Lysotracker red (scale bar: 50 μm) in (3) above.

[0104] Regarding (1) above, as shown in the live cell imaging results in Figure 12, no expression or localization of Ubl3 was observed in BV2 cells. Regarding (2) above, as shown in the immunocytochemical staining results in Figure 13, no expression of Ubl3 was observed in BV2 cells. We therefore hypothesized that BV2 cells may utilize an alternative mechanism or that Ubl3 may be degraded via lysosomal degradation.

[0105] Regarding (3) above, as is clear from the live cell imaging results shown in Figures 14 and 15, Ubl3 was expressed in BV2 cells and confirmed to be localized in the plasma membrane and multivesicular bodies. To observe the state of lysosomes in BV2 cells after CQ treatment, 100 nM Lysotracker Red was added. As is clear from the results shown in Figures 16 and 17, the Lysotracker Red signal disappeared or was very low in the CQ-treated group, whereas a very strong Lysotracker Red signal was observed in the CQ-untreated group. These results clearly confirm that UBL3 is degraded in lysosomes in BV2 cells.

[0106] Example 12 Demonstration study demonstrating that administration of AAV-UBL3 significantly improves aggregated α-synuclein pathology in mouse brain (In vivo pathological evaluation of AAV-UBL3) The propagation of aggregated protein pathology following injection of pre-formed fibrils (PFFs) was evaluated using immunohistochemistry for phosphorylated α-synuclein.

[0107] (Materials and Methods) ((Cerebral Stereotaxy)) Wild-type (C57BL / 6J) mice were anesthetized by isoflurane inhalation, and after incision of the scalp, a hole was drilled in the skull and 10 μg of PFF was injected into the striatum (coordinates: anterior-posterior 0.2 mm, lateral 2.0 mm, superior-inferior 2.8 mm). One month after injection, 10 μg of PFF was injected into the same coordinates. １０ GC (gene copy) AAV or an equal volume of PBS (2 μL) was re-injected.

[0108] (Immunofluorescence) The prepared sections were deparaffinized and rehydrated. Then, to block endogenous peroxidase activity, they were rehydrated in 10% H ２ O ２ The sections were treated with 1% bovine serum albumin (BSA) for 15 minutes. For antigen retrieval, citrate buffer (pH 6.0, Wako Pure Chemical Industries, Ltd.) was used and autoclaved at 105°C for 1 minute. The sections were blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich) at room temperature for 1 hour. Next, the sections were incubated with a primary antibody (anti-alpha-synuclein (phospho S129), Abcam) for 1 hour. Subsequently, a secondary antibody (anti-rabbit IgG) was diluted with 1% BSA-containing PBS and incubated at room temperature for 1 hour. The sections were then further incubated with biotin-based peroxidase (Vector Laboratories) for 1 hour. Staining was performed using DAB (3,3'-Diaminobenzidine, Dojindo), nuclear staining (counterstaining) was performed using hematoxylin (Sakura Finetek), and the sections were mounted in Pathomount. The prepared sections were photographed using a NanoZoomer (Hamamatsu Photonics) with a 40x objective. The results are shown in Figures 18-1 to 18-3.

[0109] (Results) Figures 18-1 to 18-3 show the results of UBL3-AAV-mediated aggregation degradation in α-synuclein-injected mouse models. As is clear from the results shown in Figures 18-1 to 18-3, compared with the PBS group, AAV-UBL3 significantly reduced aggregated protein pathology in the cerebral cortex and substantia nigra of the injected side of the mice. This suggests that overexpression of UBL3 can degrade aggregated protein pathology formed in the brain. On the other hand, no similar effect was observed in the AAV-GFP and AAV-UBL3cx groups, indicating that this effect is specific to the AAV-UBL3-treated group. These results demonstrate that AAV-UBL3 can function as a therapeutic and / or preventive agent for disorders involving denatured protein aggregation.

[0110] Example 13: Demonstration test showing that administration of AAV-UBL3 significantly reduces intracellular aggregated α-synuclein. A pathological model was constructed by inducing pre-formed fibrils (PFFs) in neurons overexpressing α-synuclein, and treatment with AAV was performed. Thioflavin S was used to specifically fluorescently label aggregated proteins, and DAPI was used for nuclear staining.

[0111] (Materials and Methods) PFF was transfected into SH-SY5Y neurons overexpressing α-syn using XtremeGene HP (Roche). 24 hours after PFF transfection, AAV was added at an MOI (number of viruses per cell) of 10,000. 24 hours after AAV treatment, the cells were stained with 0.5% Thioflavin S (Merck) and DAPI, and images were photographed and analyzed using an InCell Analyzer (GE Healthcare Life Sciences). The results are shown in Figure 19 (Scale Bar: 50 μm).

[0112] (Results) As is clear from the results shown in Figure 19, AAV-UBL3 reduced intracellular aggregated proteins, but an increase in aggregated proteins was observed in the AAV-UBL3cx (dominant negative) treatment group. These results demonstrate that AAV-UBL3 can function as a therapeutic and / or preventive agent for denatured protein aggregation disorders.

[0113] Example 14: Test to confirm that UBL3 interacts selectively (preferably specifically) with polyglutamine (polyQ)-expanded huntingtin (mHTT) and regulates its intracellular transport We confirmed that UBL3 interacts specifically with polyQ-expanded huntingtin (mHTT) and regulates its intracellular transport. We confirmed that UBL3 expression promotes extracellular secretion of mHTT and suppresses its intracellular accumulation.

[0114] (Materials and Methods) (1) Immunohistochemical Staining Tissue sections were first deparaffinized and rehydrated. Then, to block endogenous peroxidase activity, 3% H ２ O ２The sections were treated with 10 mM citrate buffer (pH 9.0) for 10 minutes. Antigen retrieval was performed by autoclaving at 105°C for 1 minute. Blocking with 1% BSA was then performed at room temperature for 1 hour. After blocking, the sections were reacted with a primary antibody, followed by a reaction with a biotinylated secondary antibody (Vector Laboratories) at room temperature for 1 hour. After the secondary antibody reaction, the sections were incubated with ABC solution (Vectastain Elite ABC Kit) for 1 hour. Color development was performed using DAB solution for 5 minutes, and counterstaining was performed with hematoxylin. After washing and dehydration, the sections were mounted, and images were acquired using a Nanozoomer 2.0-HT slide scanner.

[0115] (2) Plasmid Construction and Cell Culture For plasmid construction, a Gluc luciferase sequence was added to the N-terminus of the NGluc-UBL3 and NGluc-UBL3Δ5 plasmids, and the C-terminal "CCVIL" sequence was deleted from UBL3Δ5. nHTTpolyQ78-CGluc was prepared by inserting a CGluc sequence between the XhoI and MluI sites in codons 1-156 of the HTT gene (including HD exon 1 and 78 CAG repeats). MYC-nHTTpolyQ78 was PCR amplified, cleaved with XhoI and XbaI, and inserted into the pcDNA3-MYC vector. Cells were cultured in 10% FBS-supplemented DMEM medium at 37°C and 5% CO. ２ When the cells reached 60-80% confluence, transfection was performed using Lipofectamine 2000. Specifically, according to the manufacturer's recommended protocol, Lipofectamine 2000 diluted in Opti-MEM was mixed with the plasmid DNA and added to the cells.

[0116] (3) Gaussia princeps luciferase complementation assay. After transfection, the culture supernatant was collected and centrifuged at 1,200 rpm for 5 minutes to remove cell debris. The cells were lysed with 1% Triton X-100 and centrifuged at 13,000 rpm for 5 minutes to collect the supernatant. For luminescence measurement, coelenterazine (17 μg / mL) diluted with Opti-MEM was added, and immediate measurements were performed using a microplate reader. The luminescence value of untreated DMEM (10% FBS) was used as background and subtracted from the luminescence values ​​of all samples.

[0117] (4) Immunocytochemical Staining. Cultured cells were fixed with ice-cold 100% methanol for 5 minutes and washed three times with ice-cold PBS. After blocking with 1% BSA for 1 hour, they were incubated with primary antibodies overnight at 4°C. After washing with PBS (5 minutes x 3 times), they were incubated with secondary antibodies at room temperature for 1 hour in the dark. Nuclei were stained with DAPI and then washed again with PBS (5 minutes x 3 times). Finally, the cells were mounted with VECTASHIELD and observed using a 63x objective lens on a Leica TCS SP8 confocal microscope. All images were captured under identical imaging conditions and image processing parameters were consistent. All of these experimental procedures were reproducible in at least three independent experiments. The results are shown in Figures 20-23.

[0118] (Result 1) Figure 20 shows immunohistochemical staining images of Huntington's disease (hereinafter also referred to simply as "HD") patients and a control group. Figure 20(A) shows an overview of the striatum stained with HE (scale bar: 10 mm) and shows the overall image of the striatum region of two HD patients and a control group. Tissue atrophy was observed in the HD patient group. Figure 20(B) shows an 1C2 staining image (scale bar: 10 μm) demonstrating the accumulation of polyglutamine in the neuronal nuclei of HD patients. The brown staining visualizes the localization of polyglutamine, while no staining is observed in the control group. Figure 20(C) shows an image stained with UBL3 (scale bar: left 100 μm, right 50 μm). In the HD patient group, distinct inclusions were observed in the cytoplasm and nucleus (indicated by arrows), while in the control group, a diffuse dot-like distribution was observed. The low-magnification image on the left shows the distribution throughout the tissue, and the high-magnification image on the right shows localization in individual cells.

[0119] As is clear from the results shown in Figures 20(A) to (C), immunohistochemical staining of postmortem brain tissue (striatum) from HD patients revealed UBL3-positive inclusions in the cytoplasm and nuclei of neurons. In neurons from the control group, UBL3 was distributed in the cytoplasm as dispersed dots, whereas in HD patients, it was present as distinct inclusions. This difference in distribution pattern was consistently observed in multiple HD patient samples (n = 2, number of polyQ repeats: 50 / 22, 53 / 20), and reproducible differences were confirmed between the HD patient and control groups (n = 1).

[0120] (Result 2) The interaction between UBL3 and mHTT was quantitatively evaluated by a Gaussia princeps luciferase complementation assay. Figures 21-1 and 21-2 show the results of analyzing the interaction between UBL3 and mHTT. Figure 21-1(A) is a schematic diagram of the constructs used in the experiment, showing the structure of nHTTpolyQ78 (amino acids 1-156, containing 78 CAG repeats) and the structures of NGluc-UBL3, NGluc-UBL3Δ5, and nHTTpolyQ78-CGluc. Figure 21-1(B) shows the results of measuring luciferase activity in the culture supernatant. The Y-axis represents luminescence intensity (logarithmic scale). The mean ± standard deviation (n = 3) for each group is shown, and a significant increase in luminescence was observed in the NGluc-UBL3 / nHTTpolyQ78-CGluc group. Figure 21-1(C) shows the results of luciferase activity measurements in cell lysates, with the Y axis representing luminescence intensity (logarithmic scale). The mean ± standard deviation (n = 3) for each group is shown, demonstrating a pattern similar to that observed in the culture supernatant. Figure 21-2(D) shows the results of an immunoprecipitation experiment. In the Western blot images of Input (1%) and Co-IP (20%), both IB:MYC and IB:Flag were detected, confirming the interaction between Flag-UBL3 and MYC-nHTTpolyQ78.

[0121] As is clear from the results shown in Figures 21-1(A) to (C) and 21-2(D), significant luminescence signals were detected in both the culture supernatant and cell lysate of HEK293 cells coexpressing NGluc-UBL3 and nHTTpolyQ78-CGluc (p<0.001, n=3). This interaction was also observed in a mutant (UBL3Δ5) lacking the C-terminal cysteine ​​motif (CCVIL) of UBL3. Furthermore, immunoprecipitation experiments confirmed the direct binding of Flag-UBL3 to MYC-nHTTpolyQ78.

[0122] (Result 3) Figure 22 shows the results of quantifying intracellular and extracellular mHTT using a HiBiT assay. Here, HiBiT is an 11-amino acid peptide tag used to detect target proteins by luminescence using a photoprotein fragment (LgBiT) and a substrate that binds to it. Figure 22(A) is a schematic diagram of the HTT-PolyQ72-HiBiT construct, showing the linkage via a flexible linker (GGGGS: SEQ ID NO: 10). Figure 22(B) shows the results of luminescence measurements in the culture supernatant, with the Y-axis representing luminescence intensity. The mean ± standard deviation (n = 4) for each group is shown, and statistical significance is indicated as *: p<0.05, **: p<0.01, ***: p<0.001, and ****: p<0.0001. Figure 22(C) shows the results of luminescence measurements in cell lysates, with the Y-axis representing luminescence intensity. The mean ± standard deviation (n = 4) for each group is shown, and statistical significance is indicated. Figure 22(D) shows the relative ratio of culture supernatant to cell lysate, with the Y axis representing the relative ratio. A boxplot is used to display the distribution, demonstrating significant changes in ratio due to UBL3 coexpression.

[0123] As is clear from the results shown in Figure 22(A)-(D), in the coexpression experiment of HiBiT-tagged HTT-PolyQ72 and UBL3, the amount of mHTT in the culture supernatant was significantly increased (p<0.001) and the amount of mHTT in the cells was significantly decreased (p<0.01) compared to the control group in the presence of UBL3. On the other hand, coexpression with UBL3Δ5 significantly decreased the amount of mHTT in both the culture supernatant and cells (p<0.001). These results indicate that UBL3 has the function of promoting the extracellular secretion of mHTT. These results suggest that UBL3 may function as an inhibitor of the propagation and / or aggregation of denatured proteins.

[0124] (Result 4) Figure 23 shows the results of immunocytochemical staining. Figure 23(A) shows the results of colocalization analysis with MYC-nHTTpolyQ78. The left column shows merged images, the middle column shows anti-Flag staining (green), and the right column shows anti-MYC staining (red). DAPI nuclear staining (blue) is also included (scale bar: 10 μm). Figure 23(B) shows the results of colocalization analysis with nHTTpolyQ72-HiBiT. The left column shows merged images, the middle column shows anti-Flag staining (green), and the right column shows anti-HiBiT staining (red). DAPI nuclear staining (blue) is also included (scale bar: 10 μm). These images show representative results, and reproducibility has been confirmed in at least three independent experiments. All images were acquired under identical imaging and processing conditions. These results suggest that UBL3 may function as an inhibitor of denatured protein propagation and / or aggregation.

Claims

A substance that enhances the expression of the UBL3 gene or UBL3 protein, and Substances that enhance the activity of UBL3 protein An agent for inhibiting the propagation and / or aggregation of denatured proteins, comprising at least one selected from the group consisting of: 　 The agent according to claim 1 , wherein the substance that enhances the expression of the UBL3 gene or UBL3 protein comprises a vector containing the UBL3 gene. 　 The agent according to claim 1 , wherein the substance that enhances the activity of the UBL3 protein comprises a substance that enhances the interaction between the UBL3 protein and a denatured protein. 　 The agent according to claim 1, wherein the denatured protein is denatured α-synuclein, TDP43, tau, or huntingtin protein or amyloid β. 　 A therapeutic and / or prophylactic agent for a denatured protein aggregation disorder, comprising the agent according to claim 1. 　 The agent according to claim 5, wherein the degenerative protein aggregation disease is at least one selected from the group consisting of synucleopathy, TDP43opathy, tauopathy, Huntington's disease, and Alzheimer's disease. 　 A pharmaceutical composition comprising the agent according to claim 1 or 5. 　 A method for screening for a therapeutic and / or prophylactic agent for a denatured protein aggregation disease, the method comprising a screening step using enhanced expression of the UBL3 gene or UBL3 protein, or enhanced activity of the UBL3 protein, as an index. 　 The method according to claim 8, wherein the activity of the UBL3 protein is an activity of the UBL3 protein interacting with a denatured protein. 　 The method according to claim 8, wherein the degenerative protein aggregation disease is at least one selected from the group consisting of synucleopathy, TDP43opathy, tauopathy, Huntington's disease, and Alzheimer's disease.

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