Prophylactic or therapeutic agent for neurodegenerative disease, inhibitor of phosphorylation of tau protein, and suppressant of abnormal localization of TDP-43 protein to cytoplasm

Abstract

Provided are a prophylactic or therapeutic agent for a neurodegenerative disease, an inhibitor of phosphorylation of tau protein, and a suppressant of abnormal localization of TDP-43 protein to the cytoplasm. The prophylactic or therapeutic agent for a neurodegenerative disease, the inhibitor of phosphorylation of tau protein, and the suppressant of abnormal localization of TDP-43 protein to the cytoplasm contain, as an active ingredient, at least one substance selected from the group consisting of an HPCAL1 gene inhibiting substance, an NDST3 gene inhibiting substance, a CALN1 gene inhibiting substance, and an AAK1 gene promoting substance.

Description

Agents for the prevention or treatment of neurodegenerative diseases, inhibitors of tau protein phosphorylation, and inhibitors of abnormal localization of TDP-43 protein in the cytoplasm.

[0001] The present invention relates to a preventive or therapeutic agent for neurodegenerative diseases, a phosphorylation inhibitor of tau protein, and an inhibitor of abnormal localization of TDP-43 protein in the cytoplasm. This application claims priority under U.S. Provisional Patent Applications 63 / 778,331, 63 / 778,340, 63 / 778,343, and 63 / 778,347, filed on 26 March 2025, the contents of which are incorporated herein by reference.

[0002] Neurodegenerative diseases are diseases in which nerve cells degenerate. Among neurodegenerative diseases, progressive supranuclear palsy (PSP), tauopathy-associated frontotemporal lobar degeneration (FTLD-tau), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick's disease, Alzheimer's disease, chronic traumatic encephalopathy (CTE), and multiple system atrophy (MSA) are collectively called tauopathy, a neurodegenerative disease in which tau protein accumulates abnormally in cells, leading to nerve loss. Furthermore, among neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), TDP-associated frontotemporal lobar degeneration (FTLD-TDP), limbic-dominant senile TDP-43 encephalopathy (LATE), and Perry syndrome are collectively referred to as TAR DNA-binding Protein of 43kDa (hereinafter referred to as TDP-43)-related diseases. These are neurological diseases in which abnormal localization of the TDP-43 protein occurs within cells, resulting in nerve loss.

[0003] Tau protein is a 55-60 kDa intracellular microtubule-associated protein that promotes and stabilizes microtubule polymerization and is most abundantly expressed in the brain. Tau protein has many phosphorylation sites, and abnormally phosphorylated tau protein forms poorly soluble neurofibrillary tangles, which accumulate abnormally in nerve cells, leading to nerve loss and degeneration of the nervous system.

[0004] Based on the above, it is suggested that inhibiting the phosphorylation of tau protein may lead to an improvement in the pathology of tauopathy. However, there are no reports of approved drugs that can treat neurodegenerative diseases by inhibiting the phosphorylation of tau protein.

[0005] TDP-43 is an RNA-binding protein that is normally localized in the nucleus. TDP-43-related diseases are thought to be partly caused by the production of abnormal TDP-43 proteins, such as protein aggregates and protein fragments. When abnormal TDP-43 proteins are produced, they can easily migrate to the cytoplasm, form protein aggregates, or undergo changes in the amount and type of post-translational modifications such as phosphorylation, leading to various TDP-43 diseases. TDP-43-related diseases generally manifest as symptoms such as motor dysfunction and cognitive impairment, which can have a significant impact on quality of life (QOL). Therefore, it is suggested that suppressing the abnormal localization of TDP-43 protein in the cytoplasm could lead to the prevention or treatment of TDP-43-related diseases. However, there have been no reports of approved drugs that can treat neurodegenerative diseases by suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0006] Hippocalcin like protein 1 (hereinafter abbreviated as HPCAL1) is a protein belonging to the neuron-specific calcium-binding protein family and is involved in intracellular calcium regulation. HPCAL1 is also referred to as VILIP-3 (Visinin-like protein 3), and it has been reported that HPCAL1 is a biomarker for diagnosing central nervous system injury (Patent Document 1), and that reduced expression of VILIP-3 induces oxidative stress (Non-Patent Document 1). However, it has not been reported that inhibiting the HPCAL1 gene can prevent or treat neurodegenerative diseases.

[0007] N-Deacetylase / N-Sulfotransferase 3 (hereinafter abbreviated as NDST3) is one of the biosynthetic enzymes for heparan sulfate / heparin, and is a type II transmembrane protein present in the Golgi apparatus. NDST3 catalyzes the N-deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin. It has been reported that in tissues and cells of patients with the C9orf72 hexanucleotide repeat expansion associated with frontotemporal dementia (FTD), NDST3 is downregulated, and that deficiency of NDST3 reduces lysosomal function (Non-Patent Document 2). However, it has not been reported that inhibiting the NDST3 gene can prevent or treat neurodegenerative diseases.

[0008] Calneuron 1 (hereinafter abbreviated as CALN1) is a calcium-binding protein with high similarity to calcium-binding proteins of the calmodulin family, and is involved in intracellular calcium regulation. It has been reported that the expression level of the CALN1 gene is decreased in specimens from Alzheimer's disease patients (Non-Patent Document 3), but it has not been reported that inhibiting the CALN1 gene can prevent or treat neurodegenerative diseases.

[0009] Adapter-associated kinase 1 (hereinafter abbreviated as AAK1) is a serine / threonine kinase belonging to the Ark1 / Prk1 family. It has been reported that AAK1 is involved in neurological diseases such as schizophrenia and neuropathic pain (Non-Patent Document 4), and treatment of diseases by inhibiting the AAK1 gene has been reported for schizophrenia and neuropathic pain. Furthermore, it has been reported that in a model animal of Alzheimer's disease, cognitive function is negatively correlated with the expression level of AAK1 in the cerebral cortex and hippocampus (Non-Patent Document 5), but it has not been reported that neurodegenerative diseases can be prevented or treated by promoting the AAK1 gene.

[0010] International Publication No. WO 2015 / 157390

[0011] Frontiers in Molecular Neuroscience 2012; 6(20): 1-12 The EMBO Journal 40: e107204, 2021 J Cell Mol Med. 2022; 26: 5779-5793 Scientific Reports 2024; 14; 6723 Journal of Molecular Neuroscience 2018; 65; 179-189

[0012] The present invention provides a medicament for treating or preventing a neurodegenerative disease.

[0013] As a result of intensive studies, the present inventors have found that an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor or an AAK1 gene promoter is effective for treating or preventing a neurodegenerative disease, and have completed the present invention. The present invention includes the following embodiments.

[0014] [1] A preventive or therapeutic agent for neurodegenerative diseases, comprising as an active ingredient at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter. [2] The preventive or therapeutic agent according to [1], wherein the neurodegenerative disease is a tauopathy. [3] The preventive or therapeutic agent according to [2], wherein the tauopathy is a primary tauopathy. [4] The preventive or therapeutic agent according to [3], wherein the primary tauopathy is selected from the group consisting of progressive supranuclear palsy, tauopathy-associated frontotemporal lobar degeneration, corticobasal degeneration, argyrophilic grain dementia, and Pick's disease. [5] The preventive or therapeutic agent according to [2], wherein the tauopathy is a secondary tauopathy. [6] The preventive or therapeutic agent according to [5], wherein the secondary tauopathy is selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, and multiple system atrophy. [7] The preventive or therapeutic agent according to any one of [1] to [6], wherein the prevention or treatment of the neurodegenerative disease is by inhibition of phosphorylation of tau protein. [8] The preventive or therapeutic agent according to [1], wherein the neurodegenerative disease is a TDP-43 related disease. [9] The preventive or therapeutic agent according to [8], wherein the TDP-43 related disease is selected from the group consisting of amyotrophic lateral sclerosis, TDP-associated frontotemporal lobar degeneration, limbic system-dominant senile TDP-43 encephalopathy, and Perry syndrome.

[10] The preventive or therapeutic agent according to [8] or [9], wherein the prevention or treatment of the neurodegenerative disease is by suppression of abnormal localization of TDP-43 protein in the cytoplasm.

[11] The prophylactic or therapeutic agent according to any one of [1] to

[10] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[12] The prophylactic or therapeutic agent according to

[11] , wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene.

[13] The prophylactic or therapeutic agent according to

[11] , wherein the antibody is an antibody against the HPCAL1 gene product.

[14] The preventive or therapeutic agent according to any one of [1] to

[13] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[15] The preventive or therapeutic agent according to

[14] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[16] The preventive or therapeutic agent according to

[14] , wherein the antibody is an antibody against the NDST3 gene product.

[17] The preventive or therapeutic agent according to any one of [1] to

[16] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[18] The preventive or therapeutic agent according to

[17] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[19] The preventive or therapeutic agent according to

[17] , wherein the antibody is an antibody against the CALN1 gene product.

[20] The preventive or therapeutic agent according to any one of [1] to

[19] , wherein the AAK1 gene promoting substance is a substance containing nucleic acid.

[21] The preventive or therapeutic agent according to

[20] , wherein the substance containing nucleic acid comprises at least one selected from the group consisting of AAK1 gene enhancing nucleic acid and AAK1 gene expression vector.

[22] The preventive or therapeutic agent according to

[21] , wherein the AAK1 gene expression vector is a viral vector.

[23] The preventive or therapeutic agent according to

[21] or

[22] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[24] The preventive or therapeutic agent according to

[23] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[25] A tau protein phosphorylation inhibitor containing at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter as an active ingredient.

[26] The inhibitor according to

[25] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[27] The inhibitor according to

[26] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene.

[28] The inhibitor according to

[26] , wherein the antibody is an antibody against the HPCAL1 gene product.

[29] The inhibitor according to any one of

[25] to

[28] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[30] The inhibitor according to

[29] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[31] The inhibitor according to

[29] , wherein the antibody is an antibody against the NDST3 gene product.

[32] The inhibitor according to any one of

[25] to

[31] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[33] The inhibitor according to

[32] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[34] The inhibitor according to

[32] , wherein the antibody is an antibody against the CALN1 gene product.

[35] The inhibitor according to any one of

[25] to

[34] , wherein the AAK1 gene promoting substance is a nucleic acid-containing substance.

[36] The inhibitor according to

[35] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an AAK1 gene-enhancing nucleic acid and an AAK1 gene expression vector.

[37] The inhibitor according to

[36] , wherein the AAK1 gene expression vector is a viral vector.

[38] The inhibitor according to

[36] or

[37] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[39] The inhibitor according to

[38] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[40] An inhibitor of abnormal localization of TDP-43 protein to the cytoplasm, comprising as an active ingredient at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters.

[41] The inhibitor according to

[40] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of nucleic acid-containing substances and antibodies.

[42] The inhibitor according to

[41] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the HPCAL1 gene.

[43] The inhibitor according to

[41] , wherein the antibody is an antibody against the HPCAL1 gene product.

[44] The inhibitor according to any one of

[40] to

[43] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[45] The inhibitor according to

[44] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[46] The inhibitor according to

[44] , wherein the antibody is an antibody against the NDST3 gene product.

[47] The inhibitor according to any one of

[40] to

[46] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[48] The inhibitor according to

[47] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[49] The inhibitor according to

[47] , wherein the antibody is an antibody against the CALN1 gene product.

[50] The inhibitor according to any one of

[40] to

[49] , wherein the AAK1 gene promoting substance is a substance containing nucleic acid.

[51] The inhibitor according to

[50] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector.

[52] The inhibitor according to

[51] , wherein the AAK1 gene expression vector is a viral vector.

[53] The inhibitor according to

[51] or

[52] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[54] The inhibitor according to

[53] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[55] A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters to a subject requiring the same.

[56] The method according to

[55] , wherein the neurodegenerative disease is tauopathy.

[57] The method according to

[56] , wherein the tauopathy is a primary tauopathy.

[58] The method according to

[57] , wherein the primary tauopathy is selected from the group consisting of progressive supranuclear palsy, tauopathy-associated frontotemporal lobar degeneration, corticobasal degeneration, argyrophilic grain dementia, and Pick's disease.

[59] The method according to

[56] , wherein the tauopathy is a secondary tauopathy.

[60] The method according to

[59] , wherein the secondary tauopathy is selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, and multiple system atrophy.

[61] The method according to any one of

[55] to

[60] , wherein the prevention or treatment of the neurodegenerative disease is by inhibition of tau protein phosphorylation.

[62] The method according to

[55] , wherein the neurodegenerative disease is a TDP-43 related disease.

[63] The method according to

[62] , wherein the TDP-43 related disease is selected from the group consisting of amyotrophic lateral sclerosis, TDP-associated frontotemporal lobar degeneration, limbic-dominant senile TDP-43 encephalopathy, and Perry syndrome.

[64] The method according to

[62] or

[63] , wherein the prevention or treatment of the neurodegenerative disease is by suppressing the abnormal localization of the TDP-43 protein in the cytoplasm.

[65] The method according to any one of

[55] to

[64] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[66] The method according to

[65] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene.

[67] The method according to

[65] , wherein the antibody is an antibody against the HPCAL1 gene product.

[68] The method according to any one of

[55] to

[67] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[69] The method according to

[68] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[70] The method according to

[68] , wherein the antibody is an antibody against the NDST3 gene product.

[71] The method according to any one of

[55] to

[70] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[72] The method according to

[71] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[73] The method according to

[71] , wherein the antibody is an antibody against the CALN1 gene product.

[74] The method according to any one of

[55] to

[73] , wherein the AAK1 gene promoting substance comprises a nucleic acid-containing substance.

[75] The method according to

[74] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an AAK1 gene enhancing nucleic acid and an AAK1 gene expression vector.

[76] The method according to

[75] , wherein the AAK1 gene expression vector is a viral vector.

[77] The method according to

[75] or

[76] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[78] The method according to

[77] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[79] A method for preventing or treating a neurodegenerative disease, comprising at least one means selected from the group consisting of means for promoting the AAK1 gene, means for inhibiting the CALN1 gene, means for inhibiting the HPCAL1 gene, and means for inhibiting the NDST3 gene.

[80] The method according to

[79] , wherein the neurodegenerative disease is a tauopathy.

[81] The method according to

[80] , wherein the tauopathy is a primary tauopathy.

[82] The method according to

[81] , wherein the primary tauopathy is selected from the group consisting of progressive supranuclear palsy, tauopathy-associated frontotemporal lobar degeneration, corticobasal degeneration, argyrophilic grain dementia, and Pick's disease.

[83] The method according to

[80] , wherein the tauopathy is a secondary tauopathy.

[84] The method according to

[83] , wherein the secondary tauopathy is selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, and multiple system atrophy.

[85] The method according to any one of

[79] to

[84] , wherein the prevention or treatment of the neurodegenerative disease is by inhibition of phosphorylation of tau protein.

[86] The method according to

[79] , wherein the neurodegenerative disease is a TDP-43 related disease.

[87] The method according to

[86] , wherein the TDP-43 related disease is selected from the group consisting of amyotrophic lateral sclerosis, TDP-associated frontotemporal lobar degeneration, limbic-dominant senile TDP-43 encephalopathy, and Perry syndrome.

[88] The method according to

[86] or

[87] , wherein the prevention or treatment of the neurodegenerative disease is by suppression of abnormal localization of TDP-43 protein in the cytoplasm.

[89] The method according to any one of

[79] to

[88] , wherein the means for inhibiting the HPCAL1 gene is the administration of an HPCAL1 gene inhibitor to a subject in which inhibition of the HPCAL1 gene is necessary.

[90] The method according to

[89] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[91] The method according to

[90] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the HPCAL1 gene.

[92] The method according to

[90] , wherein the antibody is an antibody against the HPCAL1 gene product.

[93] The method according to any one of

[79] to

[92] , wherein the means for inhibiting the NDST3 gene is the administration of an NDST3 gene inhibitor to a subject in which inhibition of the NDST3 gene is required.

[94] The method according to

[93] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[95] The method according to

[94] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the NDST3 gene.

[96] The method according to

[94] , wherein the antibody is an antibody against the NDST3 gene product.

[97] The method according to any one of

[79] to

[96] , wherein the means for inhibiting the CALN1 gene is the administration of a CALN1 gene inhibitor to a subject in which inhibition of the CALN1 gene is necessary.

[98] The method according to

[97] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[99] The method according to

[98] , wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide against the CALN1 gene, siRNA, shRNA, and a vector expressing at least one of these.

[100] The method according to

[98] , wherein the antibody is an antibody against the CALN1 gene product.

[101] The method according to any one of

[79] to

[100] , wherein the means for promoting the AAK1 gene is the administration of an AAK1 gene promoting substance to a subject in which promotion of the AAK1 gene is necessary.

[102] The method according to

[101] , wherein the AAK1 gene promoting substance is a substance containing nucleic acid.

[103] The method according to

[102] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector.

[104] The method according to

[103] , wherein the AAK1 gene expression vector is a viral vector.

[105] The method according to

[104] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[106] The method according to

[105] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[107] A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters to a subject requiring the same.

[108] The method according to

[107] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[109] The method according to

[108] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene.

[110] The method according to

[108] , wherein the antibody is an antibody against the HPCAL1 gene product.

[111] The method according to any one of

[107] to

[110] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[112] The method according to

[111] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[113] The method according to

[111] , wherein the antibody is an antibody against the NDST3 gene product.

[114] The method according to any one of

[107] to

[113] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[115] The method according to

[114] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the CALN1 gene.

[116] The method according to

[114] , wherein the antibody is an antibody against the CALN1 gene product.

[117] The method according to any one of

[107] to

[116] , wherein the AAK1 gene-promoting substance is a nucleic acid-containing substance.

[118] The method according to

[117] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acids and AAK1 gene expression vectors.

[119] The method according to

[118] , wherein the AAK1 gene expression vector is a viral vector.

[120] The method according to

[118] or

[119] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[121] The method according to

[120] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[122] A method for suppressing abnormal localization of TDP-43 protein to the cytoplasm, comprising administering an effective amount of at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters to a subject requiring the same.

[123] The method according to

[122] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of nucleic acid-containing substances and antibodies.

[124] The method according to

[123] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the HPCAL1 gene.

[125] The method according to

[123] , wherein the antibody is an antibody against the HPCAL1 gene product.

[126] The method according to any one of

[122] to

[125] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[127] The method according to

[126] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[128] The method according to

[126] , wherein the antibody is an antibody against the NDST3 gene product.

[129] The method according to any one of

[122] to

[128] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[130] The method according to

[129] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[131] The method according to

[129] , wherein the antibody is an antibody against the CALN1 gene product.

[132] The method according to any one of

[122] to

[131] , wherein the AAK1 gene promoting substance is a nucleic acid-containing substance.

[133] The method according to

[132] , wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector.

[134] The method according to

[133] , wherein the AAK1 gene expression vector is a viral vector.

[135] The method according to

[133] or

[134] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[136] The method according to

[135] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[137] A cytoplasmic TDP-43 protein degradation inhibitor containing at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters as an active ingredient.

[138] The inhibitor according to

[137] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[139] The inhibitor according to

[138] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the HPCAL1 gene.

[140] The inhibitor according to

[138] , wherein the antibody is an antibody against the HPCAL1 gene product.

[141] The inhibitor according to any one of

[137] to

[140] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[142] The inhibitor according to

[141] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the NDST3 gene.

[143] The inhibitor according to

[141] , wherein the antibody is an antibody against the NDST3 gene product.

[144] The inhibitor according to any one of

[137] to

[143] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

[145] The inhibitor according to

[144] , wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[146] The inhibitor according to

[144] , wherein the antibody is an antibody against the CALN1 gene product.

[147] The inhibitor according to any one of

[137] to

[146] , wherein the AAK1 gene promoting substance comprises a substance containing nucleic acid.

[148] The inhibitor according to

[147] , wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an AAK1 gene enhancing nucleic acid and an AAK1 gene expression vector.

[149] The inhibitor according to

[148] , wherein the AAK1 gene expression vector is a viral vector.

[150] The inhibitor according to

[148] or

[149] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[151] The inhibitor according to

[150] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[152] A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter to a subject requiring the same.

[153] The method according to

[152] , wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[154] The method according to

[153] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene.

[155] The method according to

[153] , wherein the antibody is an antibody against the HPCAL1 gene product.

[156] The method according to any one of

[152] to

[155] , wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[157] The method according to

[156] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

[158] The method according to

[156] , wherein the antibody is an antibody against the NDST3 gene product.

[159] The method according to any one of

[152] to

[158] , wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.

[160] The method according to

[159] , wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

[161] The method according to

[159] , wherein the antibody is an antibody against the CALN1 gene product.

[162] The method according to any one of

[152] to

[161] , wherein the AAK1 gene promoting substance is a substance containing nucleic acid.

[163] The method according to

[162] , wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector.

[164] The method according to

[163] , wherein the AAK1 gene expression vector is a viral vector.

[165] The method according to

[163] or

[164] , wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors.

[166] The method according to

[165] , wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[0015] According to the present invention, a pharmaceutical product effective in preventing or treating neurodegenerative diseases is provided.

[0016] This graph shows the neurite length in each experimental group using healthy-derived neurons in Example 1, calculated as a ratio based on the results at the start of stress-inducing agent treatment (0 hr). The thick line shows the results for the group without stress-inducing agent, and the dashed line shows the results for the group with stress-inducing agent. This graph shows the neurite length in each experimental group using MAPT-mutated neurons in Example 1, calculated as a ratio based on the results at the start of stress-inducing agent treatment (0 hr). The thick line shows the results for the group without stress-inducing agent, and the dashed line shows the results for the group with stress-inducing agent. This is a fluorescence microscope image showing the cytoplasmic accumulation of phosphorylated tau protein in each experimental group in the evaluation of phosphorylated tau protein cytoplasmic accumulation in Example 1. This graph shows the fluorescence intensity of phosphorylated tau protein in the cytoplasmic region of each experimental group in the evaluation of phosphorylated tau protein cytoplasmic accumulation in Example 1. This graph shows the results of measuring HPCAL1 gene expression levels with and without HPCAL1 gene inhibitors in Example 2. This graph shows the results of evaluating the occurrence of neuronal cell damage by morphological analysis of cells in each experimental group in Example 2. This graph shows the results of measuring the accumulation of phosphorylated tau protein in each experimental group in Example 2 with and without the HPCAL1 gene inhibitor. This graph shows the results of measuring the NDST3 gene expression level with and without the NDST3 gene inhibitor in Example 3. This graph shows the results of evaluating the occurrence of neuronal cell damage by morphological analysis of cells in each experimental group in Example 3. This graph shows the results of measuring the accumulation of phosphorylated tau protein in each experimental group in Example 3 with and without the NDST3 gene inhibitor. This graph shows the results of measuring the CALN1 gene expression level with and without the CALN1 gene inhibitor in Example 4. This graph shows the results of evaluating the occurrence of neuronal cell damage by morphological analysis of cells in each experimental group in Example 4. This graph shows the results of measuring the accumulation of phosphorylated tau protein in each experimental group in Example 4 with and without the CALN1 gene inhibitor. This graph shows the results of measuring the AAK1 gene expression level with and without the AAK1 gene-promoting substance in Example 5.This graph shows the results of evaluating the occurrence of neuronal cell damage by morphological analysis of cells in each experimental group in Example 5. This graph shows the results of measuring the accumulation of phosphorylated tau protein in each experimental group in Example 5 with and without the AAK1 gene promoter. This graph shows the cytoplasmic / nuclear ratio of TDP-43 fluorescence intensity with and without the HPCAL1 gene inhibitor in Example 6. This graph shows the results of evaluating the occurrence of neuronal cell damage with and without the NDST3 gene inhibitor in Example 7.

[0017] It should be understood that both the above summary and the following detailed description are illustrative and descriptive only and do not limit the claimed invention. Furthermore, the section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described herein.

[0018] (Definitions) Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, organic synthesis chemistry, and medical and medicinal chemistry described herein are well known and commonly used in the art. Standard techniques can be used in the chemical synthesis and chemical analysis used herein. Where permitted, all patents, applications, published applications, and other publications, GenBank accessions and associated sequence information and other data available through databases such as the National Center for Biotechnology Information (NCBI), which are referenced throughout this disclosure, are incorporated by reference in part and in whole in the documents discussed herein.

[0019] Furthermore, this specification is filed together with a sequence listing in electronic format, and the sequence listing information contained in said electronic format is incorporated in its entirety into this specification by reference.

[0020] Unless otherwise specified, the following terms have the meanings set forth below.

[0021] Neurodegenerative diseases are diseases in which nerve cells degenerate. Among neurodegenerative diseases, progressive supranuclear palsy (PSP), tauopathy-associated frontotemporal lobar degeneration (FTLD-tau), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick's disease, Alzheimer's disease, chronic traumatic encephalopathy (CTE), and multiple system atrophy (MSA) are collectively called tauopathy, a neurodegenerative disease in which tau protein accumulates abnormally in cells, leading to nerve loss. Furthermore, among neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), TDP-associated frontotemporal lobar degeneration (FTLD-TDP), limbic-dominant senile TDP-43 encephalopathy (LATE), and Perry syndrome are collectively referred to as TDP-43-related diseases. These are neurodegenerative diseases characterized by abnormal intracellular localization of the TDP-43 protein, such as its migration from the nucleus to the cytoplasm, as well as the formation of aggregates, leading to neuronal loss. Inclusion body myositis, a muscle disease, is also included in TDP-43-related diseases.

[0022] Tauopathy is classified into primary tauopathy and secondary tauopathy. Primary tauopathy is tauopathy in which the accumulation of tau protein is the direct cause, and examples include progressive supranuclear palsy (PSP), tauopathy-associated frontotemporal lobar degeneration (FTLD-tau), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick's disease, frontotemporal dementia, globular glial tauopathy, argyrophilic grain disease, primary age-related tauopathy, and aging-related tau astrogliopathy. Secondary tauopathy refers to tauopathy caused by accumulations of other substances in addition to tau protein. Examples include Alzheimer's disease, chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), Guam Parkinsonism-dementia complex, and Guadeloupean parkinsonism.

[0023] TDP-43-related diseases include amyotrophic lateral sclerosis (ALS), TDP-associated frontotemporal lobar degeneration (FTLD-TDP), limbic-dominant senile TDP-43 encephalopathy (LATE), Perry syndrome, and inclusion body myositis, a muscle disease.

[0024] HPCAL1 is a protein belonging to the neuron-specific calcium-binding protein family and is involved in intracellular calcium regulation. Unless otherwise specified, HPCAL1 refers to the HPCAL1 protein, and the HPCAL1 gene refers to the gene that codes for the HPCAL1 protein. The HPCAL1 gene includes, for example, various splicing variants and sequence variants such as single nucleotide polymorphisms (SNPs) transcribed from the HPCAL1 gene. Furthermore, HPCAL1 also includes variant proteins translated from sequence variants.

[0025] NDST3 is one of the enzymes in the biosynthesis of heparan sulfate / heparin and is a type II transmembrane protein located in the Golgi apparatus. NDST3 catalyzes the N-deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin. Unless otherwise specified, NDST3 refers to the NDST3 protein, and the NDST3 gene refers to the gene that encodes the NDST3 protein. The NDST3 gene includes, for example, various splicing variants and sequence variants such as single nucleotide polymorphisms (SNPs) transcribed from the NDST3 gene. Furthermore, NDST3 also includes variant proteins translated from sequence variants.

[0026] CALN1 is a calcium-binding protein that is highly similar to the calmodulin family of calcium-binding proteins and is involved in intracellular calcium regulation. Unless otherwise specified, CALN1 refers to the CALN1 protein, and the CALN1 gene refers to the gene that encodes the CALN1 protein. The CALN1 gene includes, for example, various splicing variants and sequence variants such as single nucleotide polymorphisms (SNPs) transcribed from the CALN1 gene. Furthermore, CALN1 also includes variant proteins translated from sequence variants.

[0027] AAK1 is a serine / threonine kinase belonging to the Ark1 / Prk1 family. Unless otherwise specified, AAK1 refers to the AAK1 protein, and the AAK1 gene refers to the gene that codes for the AAK1 protein. The AAK1 gene includes, for example, various splicing variants and sequence variants such as single nucleotide polymorphisms (SNPs) transcribed from the AAK1 gene. Furthermore, AAK1 also includes variant proteins translated from sequence variants.

[0028] A "nucleic acid base" refers to a heterocyclic portion that can form a pair with a base of another nucleic acid.

[0029] "Nucleic acid base sequence" refers to the sequential order of nucleic acid bases that make up an oligonucleotide.

[0030] A "nucleoside" refers to a molecule in which a sugar and a nucleic acid base are linked. In certain embodiments, the nucleoside is linked to a phosphate group.

[0031] A "nucleotide" is a molecule in which a phosphate group is attached to the sugar portion of a nucleoside. Naturally occurring nucleotides have either ribose or deoxyribose as their sugar portion.

[0032] An "oligonucleotide" refers to a polymer of nucleosides in which each nucleoside and each internucleoside bond are linked independently of each other.

[0033] "Complementary" refers to the ability of a first nucleic acid and a second nucleic acid to form pairs between their nucleic acid bases. In certain embodiments, adenine is complementary to thymidine or uracil. In certain embodiments, cytosine is complementary to guanine. In certain embodiments, 5-methylcytosine is complementary to guanine.

[0034] "Completely complementary" or "100% complementary" means that every nucleic acid base in the nucleic acid sequence of the first nucleic acid has a complementary nucleic acid base in the second nucleic acid sequence of the second nucleic acid. In certain embodiments, the first nucleic acid is a modified oligonucleotide, and the target nucleic acid is the second nucleic acid.

[0035] "Modified nucleoside" means a nucleoside having a modified sugar and / or a modified nucleic acid base. "Modified oligonucleotide" means an oligonucleotide containing at least one such modified nucleoside and / or an intermodified nucleoside bond.

[0036] "Nucleoside bond" refers to a chemical bond between nucleosides, while "modified nucleoside bond" refers to any substitution or modification from a naturally occurring nucleoside bond (i.e., a 3'-5' phosphodiester nucleoside bond). Examples include, but are not limited to, phosphorothioate nucleoside bonds. "Phosphothioate nucleoside bond" refers to a nucleoside bond in which the phosphodiester bond is modified by replacing one of the non-bridged oxygen atoms with a sulfur atom.

[0037] "Modified bases" refer to any nucleic acid base other than adenine, cytosine, guanine, thymidine, or uracil. For example, 5-methylcytosine is included, but is not limited to. "Unmodified nucleic acid bases" refer to the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0038] "Sugar" or "sugar moiety" refers to either a natural sugar moiety or a modified sugar moiety. "Modified sugar" refers to a substitution or alteration from a natural sugar, such as substituted sugar moieties and bicyclic sugars. Here, "substituted sugar moiety" refers to a furanosyl other than a natural sugar in RNA or DNA, and "bicyclic sugar" refers to a furanosyl ring modified by a bridge of two different carbon atoms on the same ring. "Bicyclic nucleic acid" refers to a nucleoside or nucleotide in which the furanose moiety of the nucleoside or nucleotide contains a "bicyclic sugar".

[0039] "siRNA" is an abbreviation for small interfering RNA, a double-stranded RNA consisting of about 20 to 30 base pairs used for gene silencing by RNA interference (RNAi). "shRNA" is an abbreviation for short hairpin RNA, a hairpin-shaped RNA sequence used for gene silencing by RNA interference.

[0040] An "antibody" is a polypeptide that specifically binds to a particular antigen, and includes polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments.

[0041] The term "compound" refers to all substances formed by chemical bonding of atoms such as C, H, O, N, and S, and includes low-molecular-weight compounds, peptides, sugars, high-molecular-weight compounds, etc.

[0042] "Administration" means giving a drug to an animal, and includes, but is not limited to, administration by a medical professional and self-administration.

[0043] "Effective dose" means the amount of the HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter of the present invention that is sufficient to achieve the desired physiological outcome in an individual requiring the drug. The effective dose may vary among individuals depending on the health and physical condition of the treated individual, the taxonomic group of the treated individual, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0044] "Prevention" means delaying or preventing the onset or occurrence of a disease, disorder, or undesirable health condition, or one or more symptoms associated with such disease, disorder, or undesirable health condition, for a period ranging from a few minutes to an indefinite period. Prevention also means reducing the risk of developing a disease, disorder, or undesirable health condition.

[0045] "Treatment" means reducing, improving, slowing the progression of, or eliminating a disease, disorder, or undesirable health condition, or one or more symptoms associated with such disease, disorder, or undesirable health condition, or partially eliminating or eradicating one or more of the causes of such disease, disorder, or undesirable health condition itself.

[0046] (Specific Embodiments) The present invention provides a preventive or therapeutic agent for neurodegenerative diseases (hereinafter sometimes referred to as "the agent of the present invention") that contains as an active ingredient at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter.

[0047] The active ingredients, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, may be used individually or in combination. Combinations of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters include: combination of HPCAL1 gene inhibitor and NDST3 gene inhibitor, combination of HPCAL1 gene inhibitor and CALN1 gene inhibitor, combination of HPCAL1 gene inhibitor and AAK1 gene promoter, combination of NDST3 gene inhibitor and CALN1 gene inhibitor, combination of NDST3 gene inhibitor and AAK1 gene promoter, CA Examples include combinations of an LN1 gene inhibitor and an AAK1 gene promoter, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene promoter, combinations of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter, and combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter.

[0048] <HPCAL1> HPCAL1 is a protein belonging to the neuron-specific calcium-binding protein family and is involved in intracellular calcium regulation. Any HPCAL1 expressed in mammals such as humans and mice may be used as HPCAL1, but human HPCAL1 is preferred. Examples of nucleic acid sequences of human HPCAL1 mRNA precursor (pre mRNA) include the nucleic acid sequences represented by SEQ ID NO: 1 or 2. The nucleic acid sequence represented by SEQ ID NO: 1 is a nucleic acid sequence with GRCh38.p14 as the reference sequence, and the nucleic acid sequence represented by SEQ ID NO: 2 is a nucleic acid sequence with T2T-CHM13v2.0 as the reference sequence. Examples of sequence variants of human HPCAL1 mRNA include sequence variants containing the nucleic acid sequences represented by SEQ ID NOs: 3 to 7. Sequence variants containing the nucleic acid sequences represented by SEQ ID NOs: 3 to 7 have a common nucleic acid sequence of CDS, and the amino acid sequence of the HPCAL1 protein encoded by the sequence variants represented by SEQ ID NOs: 3 to 7 is shown in SEQ ID NO: 8.

[0049] However, since the nucleic acid sequence of the HPCAL1 gene may vary from individual to individual, the above sequence is not the only one that is permitted. As long as it encodes the HPCAL1 protein, for example, the nucleic acid sequence of human HPCAL1 may have 90%, 95%, or 98% or more identity with SEQ ID NOs: 3-7. Furthermore, as long as it has the function of the HPCAL1 protein, the amino acid sequence of human HPCAL1 protein may have 90%, 95%, or 98% or more identity with SEQ ID NO: 8. The function of the HPCAL1 protein is not particularly limited as long as it is the function of the wild-type HPCAL1 protein, but examples include the ability to take up calcium ions into cells, promotion of tau protein phosphorylation activity, promotion of TDP-43 protein translocation from the nucleus to the cytoplasm, inhibition of cytoplasmic TDP-43 degradation, and promotion of ferroptosis.

[0050] <NDST3> NDST3 is one of the heparan sulfate / heparin biosynthesis enzymes and is a type II transmembrane protein located in the Golgi apparatus. NDST3 catalyzes the N-deacetylation and N-sulfation of N-acetylglucosamine residues in heparan sulfate and heparin. Any NDST3 expressed in mammals such as humans and mice may be used, but human NDST3 is preferred. Examples of nucleic acid sequences of human NDST3 mRNA precursors (pre-mRNA) include the nucleic acid sequences represented by SEQ ID NO: 9 or 10. The nucleic acid sequence represented by SEQ ID NO: 9 is a nucleic acid sequence with GRCh38.p14 as the reference sequence, and the nucleic acid sequence represented by SEQ ID NO: 10 is a nucleic acid sequence with T2T-CHM13v2.0 as the reference sequence. Examples of human NDST3 mRNA include sequence variants containing the nucleic acid sequence represented by SEQ ID NO: 11. The amino acid sequence of the NDST3 protein encoded by the nucleic acid sequence represented by SEQ ID NO: 11 is shown in SEQ ID NO: 12.

[0051] However, since the nucleic acid base sequence of the NDST3 gene may vary from individual to individual, the above sequence is not the only one that is permitted. As long as it encodes the NDST3 protein, for example, the nucleic acid base sequence of human NDST3 may have 90%, 95%, or 98% or more identity with SEQ ID NO: 11. Also, as long as it has the function of the NDST3 protein, the amino acid sequence of human NDST3 protein may have 90%, 95%, or 98% or more identity with SEQ ID NO: 12. The function of the NDST3 protein is not particularly limited as long as it is the function of the wild-type NDST3 protein, but examples include activity that catalyzes N-deacetylation and N-sulfation of N-acetylglucosamine residues of heparan sulfate or heparin, deacetylation of α-tubulin, promotion of phosphorylation activity of tau protein, promotion of translocation of TDP-43 protein from the nucleus to the cytoplasm, and inhibition of cytoplasmic TDP-43 degradation.

[0052] <CALN1> CALN1 is a calcium-binding protein that has high similarity to calcium-binding proteins of the calmodulin family and is involved in intracellular calcium regulation. Any CALN1 expressed in mammals such as humans and mice may be used as CALN1, but human CALN1 is preferred. Examples of nucleic acid sequences of human CALN1 mRNA precursors (premRNA) include the nucleic acid sequences represented by SEQ ID NO: 13 or 14. The nucleic acid sequence represented by SEQ ID NO: 13 is a nucleic acid sequence with GRCh38.p14 as the reference sequence, and the nucleic acid sequence represented by SEQ ID NO: 14 is a nucleic acid sequence with T2T-CHM13v2.0 as the reference sequence. Examples of sequence variants of human CALN1 mRNA include sequence variants containing the nucleic acid sequences represented by SEQ ID NOs: 15 to 17. The amino acid sequence of the CALN1 variant protein encoded by the sequence variant containing the nucleic acid sequence represented by SEQ ID NO: 15 is shown in SEQ ID NO: 18. The sequence variants represented by SEQ ID NO: 16 or 17 share a common nucleic acid base sequence of the CDS, and the amino acid sequence of the CALN1 variant protein encoded by the sequence variant represented by SEQ ID NO: 16 or 17 is shown in SEQ ID NO: 19.

[0053] However, since the nucleic acid sequence of the CALN1 gene may vary from individual to individual, the above sequence is not the only one that is permitted. As long as it encodes the CALN1 protein, for example, the nucleic acid sequence of human CALN1 may have 90%, 95%, or 98% or more identity with sequence numbers 15-17. Furthermore, as long as it has the function of the CALN1 protein, the amino acid sequence of human CALN1 protein may have 90%, 95%, or 98% or more identity with sequence number 18 or 19. The function of the CALN1 protein is not particularly limited as long as it is the function of the wild-type CALN1 protein, but examples include the ability to take up calcium ions into cells, promotion of tau protein phosphorylation activity, promotion of TDP-43 protein translocation from the nucleus to the cytoplasm, and inhibition of cytoplasmic TDP-43 degradation.

[0054] <AAK1> AAK1 is expressed in the brain and heart and regulates clathrin-coated endocytosis, a process important for synaptic vesicle recycling and receptor-mediated endocytosis. Any AAK1 expressed in mammals such as humans and mice may be used as the AAK1, but human AAK1 is preferred. Examples of the nucleic acid sequences of the human AAK1 mRNA precursor (pre mRNA) include the nucleic acid sequences represented by SEQ ID NO: 20 or 21. The nucleic acid sequence represented by SEQ ID NO: 20 is a nucleic acid sequence with GRCh38.p14 as the reference sequence, and the nucleic acid sequence represented by SEQ ID NO: 21 is a nucleic acid sequence with T2T-CHM13v2.0 as the reference sequence. Examples of sequence variants of human AAK1 mRNA include sequence variants containing the nucleic acid sequences represented by SEQ ID NOs: 22 to 26. The amino acid sequences of AAK1 variant proteins encoded by sequence variants containing the nucleic acid sequences represented by SEQ ID NOs: 22 to 26 are shown in SEQ ID NOs: 27 to 31, respectively.

[0055] However, since the nucleic acid base sequence of the AAK1 gene may vary from individual to individual, the above sequence is not the only one that is permitted. As long as it encodes the AAK1 protein, for example, the nucleic acid base sequence of human AAK1 may have 90%, 95%, or 98% or more identity with sequence numbers 22-26. Also, as long as it has the function of the AAK1 protein, the amino acid sequence of human AAK1 protein may have 90%, 95%, or 98% or more identity with sequence numbers 27-31. As for the function of the AAK1 protein, there are no particular restrictions as long as it is the function of the wild-type AAK1 protein, but examples include promoting clathrin-coated vesicle formation, inhibiting the phosphorylation of tau protein, suppressing the translocation of TDP-43 protein from the nucleus to the cytoplasm, and suppressing cytoplasmic TDP-43 degradation.

[0056] <HPCAL1 gene inhibitors, NDST3 gene inhibitors, and CALN1 gene inhibitors> HPCAL1 gene inhibitors include substances that inhibit the function of HPCAL1 and substances that inhibit the expression of the HPCAL1 gene. NDST3 gene inhibitors include substances that inhibit the function of NDST3 and substances that inhibit the expression of the NDST3 gene. CALN1 gene inhibitors include substances that inhibit the function of CALN1 and substances that inhibit the expression of the CALN1 gene.

[0057] One example of the functions of HPCAL1, NDST3, and CALN1 is the promotion of tau protein phosphorylation.

[0058] Therefore, HPCAL1 gene inhibitors, NDST3 gene inhibitors, and CALN1 gene inhibitors are substances that can prevent or treat neurodegenerative diseases such as tauopathy by inhibiting the phosphorylation of tau protein.

[0059] The phosphorylation inhibitory effects of HPCAL1 gene inhibitors, NDST3 gene inhibitors, and CALN1 gene inhibitors on tau protein can be measured using the system described later. The agents of the present invention preferably reduce tau protein phosphorylation by 90% or less, more preferably by 70% or less, more preferably by 50% or less, more preferably by 30% or less, and may also be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0060] Furthermore, the functions of HPCAL1, NDST3, and CALN1 include promoting the translocation of TDP-43 protein from the nucleus to the cytoplasm, or suppressing cytoplasmic TDP-43 degradation, thereby promoting the abnormal localization of TDP-43 protein in the cytoplasm.

[0061] Therefore, HPCAL1 gene inhibitors, NDST3 gene inhibitors, and CALN1 gene inhibitors are substances that can prevent or treat neurodegenerative diseases such as TDP-43-related diseases by suppressing the translocation of TDP-43 protein from the nucleus to the cytoplasm, or by promoting the degradation of cytoplasmic TDP-43, thereby suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0062] The inhibitory effect of HPCAL1 gene inhibitors, NDST3 gene inhibitors, and CALN1 gene inhibitors on the abnormal localization of TDP-43 protein in the cytoplasm can be measured using the system described later. The agents of the present invention preferably reduce the abnormal localization of TDP-43 protein in the cytoplasm by 90% or less, more preferably by 70% or less, more preferably by 50% or less, more preferably by 30% or less, and may also be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0063] The agent of the present invention may be an agent that directly acts on at least one selected from the group consisting of HPCAL1, NDST3, and CALN1 to inhibit the above function, or it may be an agent that indirectly acts on at least one selected from the group consisting of HPCAL1, NDST3, and CALN1 to inhibit the above function.

[0064] On the other hand, inhibiting the expression of the HPCAL1 gene means reducing the amount of one or more of the HPCAL1 mRNA precursor, mRNA, and protein. Inhibiting the expression of the NDST3 gene means reducing the amount of one or more of the NDST3 mRNA precursor, mRNA, and protein. Inhibiting the expression of the CALN1 gene means reducing the amount of one or more of the CALN1 mRNA precursor, mRNA, and protein.

[0065] The inhibition of the expression of the HPCAL1 gene, NDST3 gene, and CALN1 gene can be evaluated using the expression measurement system described later. The agent of the present invention preferably reduces the amount of at least one mRNA precursor, mRNA, and / or protein selected from the group consisting of HPCAL1, NDST3, and CALN1 to 70% or less, preferably 50% or less, more preferably 40% or less, particularly preferably 30% or less, and may also be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0066] The HPCAL1 gene inhibitor, NDST3 gene inhibitor, and CALN1 gene inhibitor are not particularly limited in type, as long as they are substances that can inhibit the function of HPCAL1 and / or the expression of the HPCAL1 gene, the function of NDST3 and / or the expression of the NDST3 gene, and the function of CALN1 and / or the expression of the CALN1 gene, respectively. Examples include substances containing nucleic acids, antibodies, and compounds.

[0067] Substances containing nucleic acids that are HPCAL1 gene inhibitors, NDST3 gene inhibitors, or CALN1 gene inhibitors include, for example, antisense oligonucleotides, siRNAs, shRNAs, or vectors expressing at least one of these against HPCAL1, NDST3, or CALN1, but antisense oligonucleotides against HPCAL1, NDST3, or CALN1 are preferably used.

[0068] The antisense oligonucleotide for HPCAL1 is preferably an antisense oligonucleotide consisting of 10 to 50 bases, preferably 15 to 30 bases, and containing a continuous nucleic acid sequence of at least 8 bases that is 100% complementary to the isolength portion of the nucleic acid sequence of HPCAL1 (for example, SEQ ID NOs. 3 to 7 or nucleic acid sequences having 90%, 95%, or 98% or more identity thereto) (hereinafter referred to as the "HPCAL1 complementary nucleic acid sequence"). Here, the isolength portion means the portion that is complementary between the nucleic acid sequence of the antisense oligonucleotide and the nucleic acid sequence of HPCAL1.

[0069] The antisense oligonucleotide for HPCAL1 may have a total length nucleic acid sequence of 10 to 50 bases that includes the HPCAL1 complementary nucleic acid sequence, and may have one or more mismatched nucleic acid bases or added bases in the portion other than the HPCAL1 complementary nucleic acid sequence, and may have a total complementarity of 85% or more, 90% or more, preferably 95% or more, and more preferably 100% with respect to the equal-length portion of the HPCAL1 nucleic acid sequence (e.g., SEQ ID NOs: 3 to 7).

[0070] The antisense oligonucleotide for NDST3 is preferably an antisense oligonucleotide consisting of 10 to 50 bases, preferably 15 to 30 bases, and containing a continuous nucleic acid sequence of at least 8 bases that is 100% complementary to the isolength portion of the nucleic acid sequence of NDST3 (for example, Sequence ID No. 11 or a nucleic acid sequence having 90%, 95%, or 98% or more identity therewith) (hereinafter referred to as the "NDST3 complementary nucleic acid sequence"). Here, the isolength portion means the portion that is complementary between the nucleic acid sequence of the antisense oligonucleotide and the nucleic acid sequence of NDST3.

[0071] The antisense oligonucleotide for NDST3 may have a total length nucleic acid sequence of 10 to 50 bases that includes the NDST3 complementary nucleic acid sequence, and may have one or more mismatched nucleic acid bases or added bases in the portion other than the NDST3 complementary nucleic acid sequence, and may have a total length complementarity of 85% or more, 90% or more, preferably 95% or more, and more preferably 100% with respect to the isolength portion of the NDST3 nucleic acid sequence (e.g., Sequence ID No. 11).

[0072] The antisense oligonucleotide for CALN1 is preferably an antisense oligonucleotide consisting of 10 to 50 bases, preferably 15 to 30 bases, and containing a continuous nucleic acid sequence of at least 8 bases that is 100% complementary to the isolength portion of the nucleic acid sequence of CALN1 (for example, SEQ ID NOs. 15 to 17 or nucleic acid sequences having 90%, 95%, or 98% or more identity thereto) (hereinafter referred to as the "CALN1 complementary nucleic acid sequence"). Here, the isolength portion means the portion that is complementary between the nucleic acid sequence of the antisense oligonucleotide and the nucleic acid sequence of CALN1.

[0073] The antisense oligonucleotide for CALN1 can have a total length nucleic acid sequence of 10 to 50 bases that includes the CALN1 complementary nucleic acid sequence, and may have one or more mismatched nucleic acid bases or added bases in the portion other than the CALN1 complementary nucleic acid sequence. In terms of total length, it should have 85% or more, 90% or more, preferably 95% or more, and more preferably 100% complementarity with respect to the equal-length portion of the CALN1 nucleic acid sequence (for example, SEQ ID NOs: 15 to 17).

[0074] Furthermore, the respective percent complementarity between the nucleotide sequence of HPCAL1 and the antisense oligonucleotide that is an HPCAL1 gene inhibitor, between the nucleotide sequence of NDST3 and the antisense oligonucleotide that is an NDST3 gene inhibitor, and between the nucleotide sequence of CALN1 and the antisense oligonucleotide that is a CALN1 gene inhibitor can be routinely determined, for example, by using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0075] The antisense oligonucleotide against HPCAL1, NDST3, or CALN1 may be a modified oligonucleotide. The modified oligonucleotide may contain modified bases such as 5-methylcytosine, or at least one nucleoside constituting the oligonucleotide may contain a modified sugar. Alternatively, the internucleoside linkage may be modified.

[0076] Herein, a modified sugar refers to a sugar moiety that has been modified, and a modified oligonucleotide containing one or more such modified sugars has advantageous characteristics such as enhanced nuclease stability and increased binding affinity. It is preferred that at least one of the modified sugars has a bicyclic sugar or a substituted sugar moiety.

[0077] Examples of nucleosides containing modified sugars include 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ３ , 2'-OCH ２ CH ３ , 2'-OCH ２ CH ２ F and 2'-O(CH ２ ) ２ OCH ３ nucleosides containing the substituent. The substituent at the 2'-position may be allyl, amino, azido, thio, O-allyl, O-C １ to C １０ alkyl, OCF３ , OCH ２ F, O(CH ２ ) ２ SCH ３ , O(CH ２ ) ２ -O-N(R) ｍ ) (Caution ｎ ), O-CH ２ -C(=O)-N(R ｍ ) (Caution ｎ ) and O-CH ２ -C(=O)-N(R ｌ )-(CH ２ ) ２ -N(R) ｍ ) (Caution ｎ ) (wherein, each R ｌ , R ｍ and R ｎ These are independently H or substituted or unsubstituted C １ ~C １０ You can also choose from alkyl groups.

[0078] Examples of nucleosides containing bicyclic sugars include nucleosides containing a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the oligonucleotides provided herein include nucleosides having one or more bicyclic sugars, wherein the bridge comprises one of the following formulas: 4'-(CH ２ )-O-2'(LNA);4'-(CH ２ )-S-2';4'-(CH ２ ) ２ -O-2'(ENA);4'-CH(CH ３ )-O-2' and 4'-CH(CH ２ OCH ３ )-O-2' (and analogues thereof; see U.S. Patent No. 7,399,845); 4'-C (CH ３ ) (CH ３ )-O-2' (and their analogues; see WO2009 / 006478); 4'-CH ２ -N(OCH) ３ )-2' (and their analogues; see WO2008 / 150729); 4'-CH ２ -O-N(CH ３)-2' (See US2004-0171570); 4'-CH ２ -N(R)-O-2' (wherein R is H, C) １ ~C １２ (An alkyl or protecting group) (See U.S. Patent No. 7,427,672); 4'-CH ２ -C(H)(CH ３ )-2' (See Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH ２ -C (=CH ２ )-2' (and their analogues; see WO2008 / 154401).

[0079] In certain embodiments, the bicyclic sugar-containing nucleoside may be a nucleoside containing the sugar portion of the cross-linked artificial nucleic acid ALNA disclosed in WO2020 / 100826, for example, a nucleoside containing the sugar portion of ALNA[Ms] represented by the following general formula (I).

[0080]

[0081] [In the formula, B is a nucleic acid base which may be substituted with one or more substituents; R １ , R ２ , R ３ and R ４ Each of these C atoms may be independently substituted with a hydrogen atom or one or more substituents. １－６ It is an alkyl group; R ５ and R ６ Each of these is independently a hydrogen atom, a hydroxyl protecting group, an optionally substituted phosphate group, a phosphorus moiety, or a covalent bond to a support; m is 1 or 2; and M is a sulfonyl group substituted with an optionally substituted methyl group. A typical example of ALNA[Ms] is a nucleoside in which M is an unsubstituted methyl group-substituted sulfonyl group. The sugar portion of ALNA constituting a modified oligonucleotide has a structure represented by the following general formula (I-S).

[0082]

[0083] [In the formula, R １ , R ２ , R ３ , R ４ , M, and m are the same as those in the general formula (I) above. * represents binding to a nucleic acid base. ** represents binding to an internucleoside bond with a nucleoside adjacent to the 5' side. *** represents binding to an internucleoside bond with a nucleoside adjacent to the 3' side.

[0084] In certain embodiments, the antisense oligonucleotide as an HPCAL1 gene inhibitor, NDST3 gene inhibitor, or CALN1 gene inhibitor has a nucleic acid sequence in which at least one nucleic acid base is cytosine. In certain embodiments, at least one cytosine is the modified nucleic acid base 5-methylcytosine.

[0085] The naturally occurring nucleoside-to-nucleoside bonds in RNA and DNA are 3'-5' phosphodiester bonds. Oligonucleotides with one or more modified, i.e., non-naturally occurring, nucleoside-to-nucleoside bonds are often preferred over naturally occurring nucleoside-to-nucleoside-to-nucleoside bonds for reasons such as enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0086] Oligonucleotides having modified nucleoside bonds include nucleoside bonds that retain a phosphorus atom and nucleoside bonds that do not contain a phosphorus atom. Typical phosphorus-containing nucleoside bonds include, but are not limited to, one or more phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.

[0087] In certain embodiments, the nucleoside bonds of antisense oligonucleotides as HPCAL1 gene inhibitors, NDST3 gene inhibitors, or CALN1 gene inhibitors are all phosphorothioate nucleoside bonds.

[0088] Antisense oligonucleotides as HPCAL1 gene inhibitors, NDST3 gene inhibitors, or CALN1 gene inhibitors can be synthesized by conventional methods, for example, easily using commercially available nucleic acid synthesizers. Furthermore, ALNA[Ms], which may be included in antisense oligonucleotides, can be synthesized by the method disclosed in WO2020 / 100826.

[0089] The siRNAs for HPCAL1, NDST3, or CALN1 are double-stranded oligoRNAs having a sequence complementary to a partial sequence of the mRNA of HPCAL1, NDST3, or CALN1 (usually 20 bases or more, preferably 21 bases or more, and usually 30 bases or less, preferably 27 bases or less, more preferably 23 bases or less), and are not particularly limited as long as they can specifically recognize and cleave the transcript, thereby inhibiting the expression of HPCAL1, NDST3, or CALN1, respectively. Those skilled in the art can determine the sequences of the sense strand and antisense strand of a siRNA that can be used to inhibit the expression of human HPCAL1 based on the nucleic acid base sequences of the human HPCAL1 gene described in SEQ ID NOs: 3 to 7. Specific examples of the sense strand and antisense strand of HPCAL1 include the sense strand shown in SEQ ID NO: 32 and the antisense strand shown in SEQ ID NO: 33. Furthermore, a person skilled in the art can determine the sequences of the sense strand and antisense strand of siRNA that can be used to inhibit the expression of human NDST3, based on the nucleic acid sequence of the human NDST3 gene described in Sequence ID No. 11. Specific examples of the sense strand and antisense strand of NDST3 include the sense strand shown in Sequence ID No. 34 and the antisense strand shown in Sequence ID No. 35. Also, a person skilled in the art can determine the sequences of the sense strand and antisense strand of siRNA that can be used to inhibit the expression of human CALN1, based on the nucleic acid sequences of the human CALN1 gene described in Sequence ID Nos. 15-17. Specific examples of the sense strand and antisense strand of CALN1 include the sense strand shown in Sequence ID No. 36 and the antisense strand shown in Sequence ID No. 37. siRNA synthesis can be carried out by known methods; for example, the sense strand and antisense strand can be synthesized by DNA / RNA chemical synthesis or enzymatic synthesis, respectively, and then annealing them.

[0090] Vectors expressing at least one of antisense oligonucleotides, siRNA, or shRNA for HPCAL1, NDST3, or CALN1 include, for example, lipid particles or plasmid vectors in which nucleic acids are encapsulated in a lipid membrane, or viral vectors and non-viral vectors in which nucleic acids are encapsulated in a capsid. Examples of plasmid vectors include plasmids derived from Escherichia coli (e.g., pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (e.g., pUB110, pTP5, pC194), and plasmids derived from yeast (e.g., pSH19, pSH15). Examples of viral vectors include DNA-containing viral vectors such as adenovirus vectors and adeno-associated virus vectors, and RNA-containing viral vectors such as retroviral vectors and lentiviral vectors. The nucleic acid contains a nucleic acid base sequence including at least one nucleic acid base sequence and / or its complementary strand, selected from antisense oligonucleotides, siRNA, or shRNA. Furthermore, the nucleic acid may include a promoter base sequence, a replication origin sequence for replication in cells, and a selection marker sequence such as a drug resistance gene. Examples of promoter base sequences include, but are not limited to, sequences expressible in mammalian cells such as SV40 and CMV, sequences expressible in E. coli such as trp, lacI, and lacZ, and sequences expressible in vitro such as SP6 and T7.

[0091] Examples of the HPCAL1 gene product include mRNA transcribed from the HPCAL1 gene and HPCAL1, a protein translated from the mRNA transcribed from the HPCAL1 gene; examples of the NDST3 gene product include mRNA transcribed from the NDST3 gene and NDST3, a protein translated from the mRNA transcribed from the NDST3 gene; and examples of the CALN1 gene product include mRNA transcribed from the CALN1 gene and CALN1, a protein translated from the mRNA transcribed from the CALN1 gene. The antibody against the HPCAL1 gene product is preferably an antagonist antibody that specifically binds to HPCAL1 and inhibits the function of HPCAL1 by binding. The antibody against the NDST3 gene product is preferably an antagonist antibody that specifically binds to NDST3 and inhibits the function of NDST3 by binding. The antibody against the CALN1 gene product is preferably an antagonist antibody that specifically binds to CALN1 and inhibits the function of CALN1 by binding.

[0092] As used herein, "antibodies" include naturally occurring antibodies such as polyclonal antibodies and monoclonal antibodies, chimeric antibodies that can be produced using genetic engineering techniques, humanized antibodies and single-chain antibodies, human antibodies that can be produced using human antibody-producing transgenic animals, antibodies produced by phage display, and their binding fragments.

[0093] A binding fragment refers to a region of the antibody mentioned above, specifically, for example, F(ab') ２ Examples include Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), and dAb (single domain antibody) (Exp. Opin. Ther. Patents, Vol. 6, No. 5, pp. 441-456, 1996).

[0094] The antibody class is not particularly limited and includes antibodies having any isotype such as IgG, IgM, IgA, IgD, or IgE. Preferably, it is IgG or IgM, and more preferably IgG considering ease of purification, etc.

[0095] Polyclonal antibodies can be produced, for example, as follows: Immunosensitization is performed by subcutaneously, intramuscularly, intravenously, intra-foot, or intraperitoneally injecting an immunogen into animals such as mice, rats, hamsters, guinea pigs, goats, horses, or rabbits one to several times. Typically, immunization is performed one to five times at intervals of approximately one to 14 days from the initial immunization, and serum is obtained from the immunosensitized mammals approximately one to five days after the final immunization. While it is possible to use the serum directly as polyclonal antibodies, it is preferably isolated and / or purified by ultrafiltration, ammonium sulfate fractionation, euglobulin precipitation, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), affinity column chromatography using an anti-immunoglobulin column or protein A / G column, or a column crosslinked with the immunogen.

[0096] Monoclonal antibodies can be produced, for example, as follows: a hybridoma is prepared from antibody-producing cells obtained from immunosensitized animals such as mice, rats, or hamsters administered an immunogen, and myeloma cells that do not produce autoantibodies; the hybridoma is cloned, and a clone that produces a monoclonal antibody showing specific affinity to the immunogen used for mammalian immunization is selected.

[0097] The preparation of hybridomas (fusion cells) that secrete monoclonal antibodies can be carried out according to the method of Köhler and Milstein et al. (Nature, Vol. 256, pp. 495-497, 1975) and similar modification methods. Examples of myeloma cells used for cell fusion include mouse-derived myeloma p3 / X63-AG8.653 (653; ATCC No. CRL1580), p3 / NSI / 1-Ag4-1 (NS-1), p3 / X63-Ag8.U1 (p3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0 or BW5147, and rat-derived myeloma 210RCY3-Ag. 2.3. Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11, or CEM-T15 can be used.

[0098] Screening of hybridoma clones that produce monoclonal antibodies can be performed by culturing the hybridomas, for example, in a microtiter plate, and measuring the reactivity of the culture supernatant of the wells in which growth was observed to the immunogen used in the aforementioned immunosensitization, for example, by an enzyme immunoassay such as ELISA. Monoclonal antibodies are preferably isolated and / or purified, similar to the polyclonal antibodies described above.

[0099] Chimeric antibodies can be manufactured by referring to, for example, "Experimental Medicine (Special Supplement), Vol. 6, No. 10, 1988" and Japanese Patent Publication No. 3-73280, humanized antibodies can be manufactured by referring to, for example, Japanese Patent Publication No. 4-506458 and Japanese Patent Publication No. 62-296890, and human antibodies can be manufactured by referring to, for example, "Nature Genetics, Vol. 15, pp. 146-156, 1997", "Nature Genetics, Vol. 7, pp. 13-21, 1994", Japanese Patent Publication No. 4-504365, International Patent Application Publication WO94 / 25585, "Nature, Vol. 368, pp. 856-859, 1994", and Japanese Patent Publication No. 6-500233.

[0100] Antibody production using phage display allows for easy acquisition of antibodies such as Fab by recovering and concentrating phages with affinity for an antigen from a phage library prepared for antibody screening, for example, by biopanning. For more information on antibody production using phage display, please refer to "Nature, Vol. 348, pp. 552-554, 1990", ""Phage display a laboratory manual" In cold spring harbor laboratory press, 2001", and "Antibody Engineering - a Practical Approach, IRL Press, Oxford, 1996".

[0101] F(ab) ２ Fab' and Fab' can be produced by treating immunoglobulins with the proteolytic enzymes pepsin or papain, respectively. Fab can be produced by screening a Fab-expressing phage library in the same manner as the antibody production method using phage display described above.

[0102] The active ingredients of the agent of the present invention, namely the HPCAL1 gene inhibitor, the NDST3 gene inhibitor, or the CALN1 gene inhibitor, may be compounds that inhibit the function or expression of HPCAL1, NDST3, or CALN1, respectively. The structure of the compound is not limited as long as it inhibits the function or expression of HPCAL1, NDST3, or CALN1, and may be a small molecule compound, peptide, sugar, high molecule compound, etc. Compounds can be obtained by screening.

[0103] <AAK1 gene promoting substances> AAK1 gene promoting substances include substances that promote the function of AAK1 and substances that promote the expression of the AAK1 gene.

[0104] One of the functions of AAK1 is, for example, to inhibit the phosphorylation of tau protein.

[0105] Therefore, AAK1 gene promoters are substances that can prevent or treat neurodegenerative diseases such as tauopathy by inhibiting the phosphorylation of tau protein.

[0106] The inhibitory effect of the AAK1 gene-promoting substance on the phosphorylation of tau protein can be measured using the system described later. The agent of the present invention preferably reduces the phosphorylation of tau protein by 90% or less, more preferably by 70% or less, more preferably by 50% or less, more preferably by 30% or less, and may also be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0107] Furthermore, AAK1's functions include inhibiting the translocation of TDP-43 protein from the nucleus to the cytoplasm, or inhibiting cytoplasmic TDP-43 degradation, thereby suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0108] Therefore, AAK1 gene-promoting substances can prevent or treat neurodegenerative diseases such as TDP-43-related diseases by suppressing the translocation of TDP-43 protein from the nucleus to the cytoplasm or promoting cytoplasmic TDP-43 degradation, thereby suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0109] The inhibitory effect of the AAK1 gene-promoting substance on the abnormal localization of TDP-43 protein in the cytoplasm can be measured using the system described later. The agent of the present invention preferably reduces the abnormal localization of TDP-43 protein in the cytoplasm by 90% or less, more preferably by 70% or less, more preferably by 50% or less, more preferably by 30% or less, and may also be 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less.

[0110] The agent of the present invention may be an agent that directly acts on AAK1 to inhibit the above function, or it may be an agent that indirectly acts on AAK1 to inhibit the above function.

[0111] On the other hand, promoting the expression of the AAK1 gene means increasing the amount of one or more of the AAK1 mRNA precursor, mRNA, and protein.

[0112] The enhancement of AAK1 gene expression can be evaluated using the expression measurement system described later. The agent of the present invention preferably increases the amount of AAK1 mRNA precursor, mRNA, and / or protein by 1.2 times or more, more preferably by 1.5 times or more, and particularly preferably by 2 times or more, compared to when the agent is not added or when a negative control is added.

[0113] AAK1 gene-promoting substances are not particularly limited in type as long as they can promote the function of AAK1 and / or the expression of the AAK1 gene, but examples include substances and compounds containing nucleic acids.

[0114] One embodiment of the agent of the present invention contains an AAK1 gene-promoting substance as an active ingredient. The promoting substance has one or more characteristics that increase the amount and / or activity of a predetermined translation product in various target applications, including cells, specimens, and living organisms. In the following description, the substance will also be simply referred to as the "promoting substance." In the present invention, the promoting substance directly or indirectly promotes gene expression from a predetermined nucleic acid. This makes it possible to increase the intracellular amount of at least the predetermined translation product. In this specification, "gene expression" or "gene expression" includes not only the synthesis (production) of translation products based on information of a predetermined nucleic acid base sequence, but also the synthesis (production) of transcripts. In detail, the promoting substance of the present invention promotes at least one selected from the production of transcripts from nucleic acids encoding a predetermined protein, the production of translation products, the function of transcripts, and the function of translation products within the cell. Specific examples of nucleic acids to be promoted may include polymers of deoxyribonucleotides such as endogenous DNA such as genomic DNA, introduced DNA, and synthetic DNA such as complementary DNA; and polymers of ribonucleotides such as RNA. Examples of such promoting substances include, but are not limited to, various organic compounds (including so-called low molecular weight compounds), nucleic acids, proteins, lipids, and combinations thereof. The promoting substance in this disclosure may also be the translation product to be increased itself. The above-mentioned promoting substances may be used individually or in combination of two or more. Preferred embodiments of the promoting substances will be described later.

[0115] Preferably, the transcript is any RNA synthesized using DNA as a template, regardless of whether RNA processing is performed. Specific examples of RNA include various ribonucleotide polymers such as mRNA precursors, mature mRNA, and non-coding RNAs such as microRNA (miRNA). In one embodiment, the above-mentioned RNA is preferably mRNA precursor and / or mature mRNA. Increasing the expression level of transcripts such as mRNA increases the amount of translation template, thus contributing to an increase in the expression level of the target protein. Examples of translation products include proteins produced by translation from any mRNA, regardless of whether post-translational modifications are performed. These transcripts and translation products may each be, for example, wild-type products transcribed and translated from a given DNA, or they may be various splicing variants, sequence variants such as base substitutions (including SNPs), and non-wild-type proteins translated from them. The various DNAs, transcripts, and translation products described above may each be isolated by one or more known extraction or purification steps, as needed.

[0116] Preferably, the transcript is capable of producing a protein with wild-type or equivalent function. Similarly, the translation product is preferably a protein with wild-type or equivalent function. Producing these products facilitates the maintenance or normalization of the cell's inherent functions, further reducing the occurrence or progression of cell damage. As a result, it can contribute more effectively to the treatment or prevention of disease. To achieve the above-mentioned preferred embodiments, methods include, but are not limited to, introducing foreign nucleic acids encoding the target protein or promoting splicing of the target protein into translatable mature mRNA, as shown in the examples described later. To determine whether the generated translation product has function equivalent to the wild-type, for example, the enzyme activity measured by a known method can be compared with the activity of the wild-type translation product.

[0117] In this specification, "enhancement" means at least one of the following: an increase in the abundance (e.g., expression level) of a transcript and / or translation product from a given gene, and an increase in the function (e.g., activity) of the translation product, in the presence of any substance. For example, enhancement can be determined if the expression level of the transcript or translation product when the substance under evaluation is brought into contact with or exposed to a given target is greater than the expression level of the transcript or translation product in the absence of the substance under evaluation. In addition to or instead of this, enhancement can also be determined by an increase in the activity of the protein itself or an enzyme within the cell. The criteria for determining whether enhancement has occurred may be, for example, based on the magnitude of the numerical values ​​of the measured values ​​obtained by any measurement method, based on the magnitude of the arithmetic mean, geometric mean, or median calculated from the measured values ​​or their ratios, or by whether there is a statistically significant difference. When evaluating based on the ratio of measured values, for example, if the ratio of the measured value R2 in the experimental group being evaluated (R2 / R1) to the reference measured value or reference value R1 is, for example, 1.05 times or more, for example, 1.10 times or more, for example, 1.30 times or more, for example, 1.50 times or more, or for example, 2.00 times or more, then it can be determined that the process has been promoted.

[0118] Examples of methods for promoting transcript production include one or more actions such as promoting transcription from DNA to mRNA precursors, inhibiting the degradation of mRNA precursors, inhibiting the degradation of mature mRNA, or regulating or activating RNA processing that forms mature mRNA from mRNA precursors. Examples of methods for promoting translation product production include one or more actions such as promoting translation from mature mRNA or inhibiting the degradation of proteins produced through translation.

[0119] The expression level of transcripts can be measured using various measurement methods such as PCR, microarrays, and RNA sequencing, using a sample such as cultured cells, living organisms, or specimens collected from such organisms. These measurement methods are preferably quantifiable. If necessary, the transcripts may be extracted from the sample, or complementary DNA (cDNA) may be synthesized using the transcripts as a template via reverse transcription, and these may be used for the above-mentioned measurements. The expression level or activity of translation products can be measured using various measurement methods such as ELISA, Western blotting, flow cytometry, immunohistochemistry, mass spectrometry, intracellular or in vivo accumulation of substrates with added fluorescent or radioactive substances, and in vitro activity measurement using substrates, using a sample such as cultured cells, living organisms, or specimens collected from such organisms. These measurement methods are preferably quantifiable. If necessary, the translation products may be extracted from the sample, and the extract may be used for the above-mentioned measurements.

[0120] The HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, and AAK1 gene promoter of the present invention are preferably substances that inhibit the phosphorylation of tau protein or substances that suppress the abnormal localization of TDP-43 protein in the cytoplasm. Tau protein is a 55-60 kDa intracellular microtubule-associated protein that promotes and stabilizes microtubule polymerization and is most abundantly expressed in the brain. Tau protein has many sites that undergo phosphorylation, and abnormally phosphorylated tau protein forms poorly soluble neurofibrillary tangles, which accumulate abnormally in nerve cells, leading to nerve loss and degeneration of the nervous system. As one embodiment of the HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter of the present invention, it is preferable that the substance reduces the intracellular amount of phosphorylated tau protein. As another embodiment of the promoter, it is preferable that the substance at least reduces the amount of phosphorylated protein accumulated in cells. These embodiments make it possible to improve the condition of nerve cells by, for example, inhibiting the phosphorylation of tau protein within cells, thereby suppressing the accumulation of phosphorylated tau protein in nerve cells. In another embodiment, an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, or an AAK1 gene promoter is a substance that directly or indirectly promotes the production and / or activity of a translation product whose translation product is a protein having tau protein phosphorylation inhibitory activity. This makes it possible to reduce the amount of phosphorylated tau protein.

[0121] To reduce phosphorylated tau protein in cells, for example, this can be done by inhibiting the production of transcripts or translation products of the HPCAL1 gene, NDST3 gene, or CALN1 gene, or by promoting the production of transcripts or translation products of the AAK1 gene. For example, the above-mentioned HPCAL1 gene inhibitor, NDST3 gene inhibitor, or CALN1 gene inhibitor may inhibit the production of the target translation product by reducing the expression level of transcripts of the HPCAL1 gene, NDST3 gene, or CALN1 gene, respectively, or by controlling the expression of other genes that control the expression level of the target translation product. Alternatively, for example, the above-mentioned AAK1 gene promoter may promote the production of the translation product by promoting the translation of the AAK1 gene. Alternatively, for example, the above-mentioned AAK1 gene promoter may promote the production of the target translation product by increasing the expression level of the AAK1 gene transcript or by controlling the expression of other genes that control the expression level of the target translation product. In any case, these embodiments make it possible to improve the state of nerve cells, for example, by inhibiting the phosphorylation of intracellular tau protein.

[0122] The tau protein described above can be divided into four regions based on its function: the N-terminal region, the proline-rich domain (PRD), the microtubule-binding domain (MBD), and the C-terminal region. The human tau protein gene (hereinafter also referred to as MAPT) has 16 exons, and exons 2, 3, and 10 are alternatively spliced, resulting in six variants. The nucleic acid sequences of these six variants are shown in SEQ ID NOs. 38 to 43. The amino acid sequences of the tau protein variants encoded by the nucleic acid sequences represented in SEQ ID NOs. 38 to 43 are shown in SEQ ID NOs. 44 to 49, respectively. The tau protein has 85 phosphorylation sites in total, consisting of serine, threonine, and tyrosine residues. In the present invention, there are no particular restrictions on the phosphorylation sites of the tau protein as long as they are sites that can be phosphorylated. For example, in the amino acid sequence represented by Sequence ID No. 44, examples include the 46th serine (Ser) residue, the 181st threonine (Thr) residue, the 202nd serine residue, the 205th threonine residue, the 217th threonine residue, the 231st threonine residue, the 235th serine residue, the 396th serine residue, the 400th serine residue, the 403rd threonine residue, and the 404th serine residue. To determine whether or not the phosphorylation of the tau protein has been inhibited, for example, methods such as ELISA, Western blotting, flow cytometry, or immunohistochemistry can be used to determine the increase or decrease in the amount of phosphorylated tau protein in response to exposure to an AAK1 gene promoter, a CALN1 gene inhibitor, an HPCAL1 gene inhibitor, or an NDST3 gene inhibitor. The phosphorylated tau protein used in this determination is preferably the phosphorylated tau protein in which the Ser202 and Thr205 residues are phosphorylated, as observed in PSP patients. Furthermore, this determination is preferably performed using a system in which bafilomycin is added to nerve cells as a stress inducer to promote the phosphorylation of tau protein. Bafilomycin can promote the phosphorylation of tau protein by suppressing the acidification of lysosomes in cells, thereby inhibiting the degradation of tau protein and promoting the accumulation of tau protein.

[0123] TDP-43 is an RNA-binding protein, and normally most of these proteins are localized in the nucleus. Disruption of the nuclear retention mechanism for TDP-43 protein, disruption of the cytoplasmic TDP-43 degradation mechanism, or the generation of abnormal proteins such as aggregates or fragments can lead to abnormal intracellular localization of TDP-43 protein, such as migration from the nucleus to the cytoplasm, or aggregate formation, resulting in neuronal loss and degeneration of the nervous system. As one embodiment of the HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter of the present invention, it is preferable that the substance suppresses the migration of TDP-43 protein from the nucleus to the cytoplasm, or promotes cytoplasmic TDP-43 degradation, thereby reducing the abnormal localization of TDP-43 protein in the cytoplasm. Another embodiment of the promoting substance is, for example, a substance that inhibits the translocation of TDP-43 protein from the nucleus to the cytoplasm, or promotes the degradation of cytoplasmic TDP-43, thereby at least reducing the abnormal localization of TDP-43 protein in the cytoplasm within the cell. These embodiments make it possible to improve the state of nerve cells by, for example, inhibiting the translocation of TDP-43 protein from the nucleus to the cytoplasm, or by promoting the degradation of cytoplasmic TDP-43, thereby suppressing the abnormal localization of TDP-43 protein in the cytoplasm. Another embodiment is an HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoting substance, which is a substance whose translation product is a protein that has an inhibitory effect on the abnormal localization of TDP-43 protein in the cytoplasm, and which directly or indirectly promotes the production and / or activity of said translation product. This reduces the amount of TDP-43 protein in the cytoplasm and suppresses the abnormal localization of TDP-43 protein in the cytoplasm.

[0124] To suppress the abnormal localization of TDP-43 protein in the cytoplasm, for example, this can be done by inhibiting the production of transcripts or translation products of the HPCAL1 gene, NDST3 gene, or CALN1 gene, or by promoting the production of transcripts or translation products of the AAK1 gene. For example, the above-mentioned HPCAL1 gene inhibitor, NDST3 gene inhibitor, or CALN1 gene inhibitor may inhibit the production of the target translation product by reducing the expression level of transcripts of the HPCAL1 gene, NDST3 gene, or CALN1 gene, respectively, or by controlling the expression of other genes that control the expression level of the target translation product. Alternatively, for example, the above-mentioned AAK1 gene promoter may promote the production of the translation product by promoting the translation of the AAK1 gene. Alternatively, for example, the above-mentioned AAK1 gene promoter may promote the production of the target translation product by increasing the expression level of transcripts of the AAK1 gene or by controlling the expression of other genes that control the expression level of the target translation product. In any case, these embodiments make it possible to improve the state of nerve cells by suppressing, for example, the translocation of TDP-43 protein from the nucleus to the cytoplasm, thereby suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0125] TDP-43 is a protein encoded by the TARDBP gene. The human wild-type TDP-43 protein is a nucleoprotein consisting of 414 amino acids, primarily localized in the nucleus. As an example, the mRNA sequence of human wild-type TDP-43 is shown in Sequence ID No. 50 (GenBank accession number: NM_007375.4). The amino acid sequence of human wild-type TDP-43 protein is shown in Sequence ID No. 51 (GenBank accession number: NP_031401.1). In TDP-43-related diseases, the production of abnormal TDP-43 proteins is considered to be one of the causes. Abnormal proteins typically exhibit characteristics such as increased translocation to the cytoplasm, increased tendency to form protein aggregates, and altered amounts and / or types of post-translational modifications such as phosphorylation. Thus, abnormal TDP-43 proteins often have properties that differ from those of the wild-type. Examples of abnormal TDP-43 proteins that may contribute to disease onset and progression include, but are not limited to, the N-terminal fragment of the human wild-type TDP-43 protein (SEQ ID NO: 52; see Shenouda et al., Front Neurosci. 2022; 16:868556.), the C-terminal fragment protein consisting of amino acid residues from the 209th to 414th N-terminus of the human wild-type TDP-43 protein (SEQ ID NO: 53), the C-terminal fragment protein consisting of amino acid residues from the 90th to 414th N-terminus of the human wild-type TDP-43 protein (SEQ ID NO: 54), the C-terminal fragment protein consisting of amino acid residues from the 170th to 414th N-terminus of the human wild-type TDP-43 protein (SEQ ID NO: 55), the C-terminal fragment protein consisting of amino acid residues from the 174th to 414th N-terminus of the human wild-type TDP-43 protein (SEQ ID NO: 56), and amino acid variants in the wild-type TDP-43 protein (see SEQ ID NO: 51).Examples of amino acid variants of the TDP-43 protein include G294A (a variant in SEQ ID NO: 51 in which glycine at position 294 is mutated to alanine), G298S (a variant in SEQ ID NO: 51 in which glycine at position 298 is mutated to serine), A315T (a variant in SEQ ID NO: 51 in which alanine at position 315 is mutated to threonine), M337V (a variant in SEQ ID NO: 51 in which methionine at position 337 is mutated to valine), Q343R (a variant in SEQ ID NO: 51 in which glutamine at position 343 is mutated to arginine), A382T (a variant in SEQ ID NO: 51 in which alanine at position 382 is mutated to threonine), G384R (a variant in SEQ ID NO: 51 in which glycine at position 384 is mutated to arginine), and proteins having point mutations in combinations thereof.

[0126] In the present invention, the AAK1 gene promoting substance is preferably a substance containing nucleic acid. More specifically, the AAK1 gene promoting substance is preferably composed of one or more selected from an expression-enhancing nucleic acid and an expression vector. The expression-enhancing nucleic acid means a nucleic acid for expressing the target gene, and is preferably an introduced nucleic acid. In the present invention, the expression-enhancing nucleic acid is preferably a nucleic acid for promoting the expression of the target gene. The target of gene expression promotion is, for example, an endogenous nucleic acid (e.g., genomic DNA) that codes for one or more of the above-mentioned AAK1 proteins, or an introduced nucleic acid. The above-mentioned promoting substances may be used alone or in combination of multiple types. By including such substances, the phosphorylation of tau protein can be inhibited, thereby inhibiting the accumulation of phosphorylated tau protein in nerve cells, and as a result, neurodegenerative diseases can be prevented or treated. Furthermore, by including such substances, the translocation of TDP-43 protein from the nucleus to the cytoplasm can be suppressed, thereby inhibiting the abnormal localization of TDP-43 protein in the cytoplasm, and as a result, neurodegenerative diseases can be prevented or treated.

[0127] Examples of expression-enhancing nucleic acids include, but are not limited to, one or more nucleic acids selected from antisense nucleic acids, non-coding RNAs, and small molecule activating RNAs. These expression-enhancing nucleic acids have the function of enabling the expression of a target gene in a cell and / or promoting the expression of said gene in a cell. This makes it possible to increase the amount of the target translation product in the cell. These nucleic acids can be obtained or manufactured by screening, for example, a known method. In the present invention, it is preferable that the expression-enhancing nucleic acid has fewer bases than the nucleic acid contained in the expression vector described later. In the present invention, it is preferable that the expression-enhancing nucleic acid has a base count in the range of, for example, 5 to 300 bases per strand, and for example, in the range of 10 to 100 bases per strand.

[0128] Antisense nucleic acids used as expression-enhancing nucleic acids include, for example, (a) nucleic acids that regulate RNA processing, (b) nucleic acids that are partially or fully complementary to miRNA, and (c) nucleic acids that are partially or fully complementary to Natural Antisense Script (NAT). These antisense nucleic acids may bind complementaryly to the entire length of the target nucleic acid, or complementaryly to a portion of the sequence of the target nucleic acid.

[0129] Embodiment (a) above promotes RNA processing such as splicing by complementaryly binding to splicing-involved sequences in mRNA precursors to produce desired mature mRNA (e.g., mRNA capable of translating the full length of wild-type protein). This contributes to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). Embodiment (b) above inhibits the function of miRNA by complementaryly binding to miRNA. miRNA is generally a type of nucleic acid that binds complementaryly to the 3'UTR region of mRNA. Therefore, miRNA works to suppress the expression of the target gene by increasing the degradation of mRNA or suppressing translation from mRNA. This embodiment weakens or eliminates the function of miRNA by using an antisense nucleic acid that binds complementaryly to miRNA, thereby releasing the suppression of gene expression. This promotes mRNA production and translation from mRNA, thereby contributing to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). Embodiment (c) above binds complementaryly to NAT and inhibits the function of NAT. NAT is a type of nucleic acid that is produced in cells and binds complementaryly to any region of a specific mRNA. In other words, NAT increases the degradation of mRNA or suppresses translation from mRNA. This embodiment weakens or eliminates the function of NAT by a mechanism similar to that of embodiment (b) above, thereby releasing the suppression of gene expression. This promotes mRNA production and translation from mRNA, contributing to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins).

[0130] In addition to the above, antisense nucleic acids used as expression-enhancing nucleic acids include, for example, nucleic acids that can complementarily bind to regions containing various sites in mRNA, such as the transcription inhibitory element (TIE), upstreamORF (uORF), immature stop codon (PTC), and guanine and adenine repeat sequences. These antisense nucleic acids can contribute to suppressing mRNA degradation, promoting translation from mRNA, or promoting the production of mRNA capable of generating wild-type proteins or proteins with equivalent function. This contributes to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). It is preferable that these expression-enhancing nucleic acids contribute to increased production of the target wild-type protein, as this allows the wild-type protein to perform its inherent function more easily and improves the clearance of abnormal proteins.

[0131] Other antisense nucleic acids used as gene expression-enhancing nucleic acids include, for example, nucleic acids that bind complementaryly to regulatory RNA that contributes to gene expression repression, and nucleic acids that bind complementaryly to the recognition sequence of RNA-binding proteins that destabilize mRNA. In this case, the function of RNA or protein that acts to repress expression is suppressed, thus releasing the repression of gene expression. This promotes mRNA production and translation from mRNA, contributing to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins).

[0132] The number of bases in an antisense nucleic acid used as an expression-enhancing nucleic acid is typically 10 to 30 bases per strand, preferably 14 to 25 bases per strand. In any of the above embodiments, the antisense nucleic acid used as an expression-enhancing nucleic acid has at least 75%, 80%, 85%, 90%, 95%, or 100% complementarity in the sites homologous (identical) to the complementary strand of the target nucleic acid base sequence. For example, when the base sequence of an antisense nucleic acid is 20 bases, the nucleic acid base sequence of the antisense nucleic acid may not have any deletions, substitutions, or insertions compared to the nucleic acid base sequence in the complementary strand of the target nucleic acid base sequence, or, for example, 1 to 5 (or 1 to 4, 1 to 3, 1 to 2, or 1) nucleic acid bases may be deleted, substituted, or inserted. Such nucleic acids can be screened, designed, and obtained, for example, by known methods.

[0133] Non-coding RNAs include nucleic acids such as regulatory RNA and SINE element-containing transcription promoter (SINEUP). Regulatory RNA is an RNA that promotes transcription from promoters incorporated into endogenous DNA such as genomic DNA or introduced nucleic acids, thereby promoting transcription from target nucleic acids to mRNA. This can promote the production of target translation products (e.g., LRSAM1 protein). SINEUP is an RNA with a SINE factor sequence and is a polyribonucleotide having a domain that promotes protein translation and a domain with a sequence complementary to mRNA. By using it, translation from mRNA is promoted (i.e., mainly contributes to increased production of translation products). The number of bases of non-coding RNA can be appropriately set depending on its type and function.

[0134] Small activated RNA (saRNA) is a nucleic acid complementary to some or all of the base sequences of the promoter sequence in genomic DNA. This promotes translation from mRNA and contributes to increased expression of the target gene (specifically, increased production of transcripts such as mRNA and / or translation products such as proteins). The number of bases in saRNA is typically 10 to 30 per strand, preferably 18 to 24 per strand. SaRNA is typically single-stranded or double-stranded RNA.

[0135] When the AAK1 gene promoting substance includes an expression-enhancing nucleic acid, the expression-enhancing nucleic acid can be, for example, single-stranded or double-stranded, as long as the effects of the present invention are achieved. The structural framework of these nucleic acids may be, for example, deoxyribonucleotides, ribonucleotides, non-nucleotides containing bases such as pyrrolidine or piperidine, or combinations thereof. The structural units of the nucleic acid in the expression-enhancing nucleic acid may be native or non-native, as long as the effects of this disclosure are achieved. Examples of non-native forms include nucleic acids in which sugars, bases, or atoms or molecules constituting the nucleoside bond in the nucleotide are substituted, modified, or deleted from the native nucleotide.

[0136] In the present invention, the AAK1 gene promoting substance includes, for example, an expression vector configured to express the AAK1 gene. More specifically, the promoting substance includes an expression vector that contains a nucleic acid comprising a base sequence encoding a protein that promotes AAK1 expression, and is capable of expressing AAK1 in cells. Such an expression vector can express the target transcript and / or translation product in a subject and increase the amount of the translation product in various environments such as in vivo, in vitro, or ex vivo. This can lead to beneficial effects such as maintaining or improving the function of nerve cells, inhibiting the phosphorylation of tau protein, and suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0137] In this specification, "expression vector" means a vector comprising a nucleic acid including a promoter base sequence and a first base sequence operably linked to the promoter base sequence, capable of generating a new nucleic acid or protein in a cell or in vitro based on the information of the first base sequence. The first base sequence may, for example, be configured to generate mRNA encoding a predetermined protein, or to generate the expression-enhancing nucleic acid described above. In other words, the expression-enhancing nucleic acid itself is not included in the "expression vector". The nucleic acid constituting the expression vector may further include, for example, a replication origin sequence for replication in a cell, or a selection marker sequence such as a drug resistance gene, in its base sequence. Examples of promoter base sequences include, but are not limited to, sequences expressible in mammalian cells such as SV40 and CMV, sequences expressible in E. coli such as trp, lacI, and lacZ, and sequences expressible in vitro such as SP6 and T7.

[0138] The type of expression vector is not particularly limited and includes, for example, lipid particles or plasmid vectors in which nucleic acids are encapsulated in a lipid membrane, or viral vectors and non-viral vectors in which nucleic acids are encapsulated in a capsid. Examples of viral vectors include DNA-containing viral vectors such as adenovirus vectors and adeno-associated virus vectors, and RNA-containing viral vectors such as retroviral vectors and lentiviral vectors. Examples of polynucleotide structural units contained in the viral vector include deoxyribonucleotide skeletons or ribonucleotide skeletons. It is preferable that each of these skeletons is in its natural form and does not contain modified nucleic acids or atomic substitutions.

[0139] When an expression vector described above is configured to produce a predetermined protein, the produced protein may or may not contain mutations in its amino acid sequence, such as deletions, substitutions, or additions of amino acids, as long as it possesses the intended function. The identity between the produced protein or the amino acid sequence of the protein and the amino acid sequence of the protein or the corresponding wild-type protein can be, for example, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. When nucleic acids are introduced externally into cells or subjects for the purpose of producing a predetermined protein, the identity of the nucleic acid base sequence can be determined based on the designed nucleic acid base sequence, and it is acceptable that other mutations inevitably occur during the transcription and / or translation process within the cell.

[0140] In the present invention, the AAK1 gene promoting substance may include a substance that promotes the function of the translation product. One or more such promoting substances may be included in the composition. By including such promoting substances, the expression level or activity of proteins that have been reduced or may be reduced by disease can be controlled to increase, thereby effectively treating or preventing the disease. Examples of substances that promote the function of the translation product include, but are not limited to, allosteric regulators of proteins and stabilizers of post-translational modifications.

[0141] The AAK1 gene-promoting substances described above can each be independently synthesized or produced, for example, by methods known in the present art.

[0142] The AAK1 gene-promoting substance, which is the active ingredient of the agent of the present invention, may be a compound that promotes the function or expression of AAK1. The compound's structure is not limited as long as it promotes the function or expression of AAK1, and it may be a low-molecular-weight compound, peptide, sugar, high-molecular-weight compound, etc. The compound can be obtained by screening.

[0143] <Methods for evaluating or screening HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters> Any method that can verify the inhibition of intracellular expression or function of the HPCAL1, NDST3, or CALN1 gene inhibitors is acceptable for evaluating or screening (selecting) HPCAL1 gene inhibitors, NDST3 gene inhibitors, or CALN1 gene inhibitors, respectively. Any method that can verify the promotion of intracellular expression or function of the AAK1 gene is acceptable for evaluating or screening (selecting) AAK1 gene promoters. Specifically, for example, the in vitro and in vivo verification methods shown below can be used.

[0144] In the evaluation or screening of HPCAL1 gene inhibitors, NDST3 gene inhibitors, or CALN1 gene inhibitors, when HPCAL1 gene inhibition is used as an indicator, one method is to add the HPCAL1 gene inhibitor or a candidate substance to cells or tissues expressing the HPCAL1 gene, measure the expression level of HPCAL1 mRNA or protein, and compare it with the condition when the inhibitor or candidate substance is not added or when a negative control is added. When NDST3 gene inhibition is used as an indicator, one method is to add the NDST3 gene inhibitor or a candidate substance to cells or tissues expressing the NDST3 gene, measure the expression level of NDST3 mRNA or protein, and compare it with the condition when the inhibitor or candidate substance is not added or when a negative control is added. When using CALN1 gene inhibition as an indicator, one method involves adding a CALN1 gene inhibitor or a candidate substance to cells or tissues expressing the CALN1 gene, measuring the expression levels of CALN1 mRNA or protein, and comparing these levels with those when the inhibitor or candidate substance is not added or when a negative control is added.

[0145] In evaluating or screening AAK1 gene-promoting substances, when AAK1 gene promotion is used as an indicator, one method involves adding the promoting substance or a candidate substance to cells or tissues expressing the AAK1 gene, measuring the expression levels of AAK1 mRNA or protein, and comparing these levels with those when the promoting substance or candidate substance is not added or when a negative control is added.

[0146] Cells expressing the HPCAL1 gene, NDST3 gene, CALN1 gene, or AAK1 gene are not particularly limited as long as they express the HPCAL1 gene, NDST3 gene, CALN1 gene, or AAK1 gene, respectively. For example, examples include cells that have been induced into human induced pluripotent stem cells (iPS cells) by conventional methods from MAPT mutant cells, which are cells with a PSP risk mutation in the MAPT gene, or from cells derived from healthy individuals, and then differentiated into excitatory neurons (hereinafter, excitatory neurons will also be simply referred to as neurons) from each iPS cell by conventional methods.

[0147] Alternatively, an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, or an AAK1 gene promoter may be administered to a non-human animal, and the expression levels of the HPCAL1 gene, NDST3 gene, CALN1 gene, and AAK1 gene may be measured in the nerve tissue of the animal, or an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, or an AAK1 gene promoter may be administered to isolated nerve tissue, and the expression levels of the HPCAL1 gene, NDST3 gene, CALN1 gene, and AAK1 gene may be measured in the nerve tissue.

[0148] By measuring the expression levels of the HPCAL1 gene, NDST3 gene, CALN1 gene, or AAK1 gene in in vitro or in vivo HPCAL1 gene expression levels, respectively, it is possible to screen for or evaluate HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters.

[0149] Furthermore, there are no particular restrictions on the method of contacting HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters with HPCAL1 gene-expressing cells, NDST3 gene-expressing cells, CALN1 gene-expressing cells, or AAK1 gene-expressing cells, respectively. However, if the HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter is a nucleic acid, methods commonly used to introduce nucleic acids into cells, such as lipofection, electroporation, or gymnosis, can be used.

[0150] The intracellular mRNA expression levels of HPCAL1, NDST3, CALN1, or AAK1 can be measured by various methods known in the art. Specifically, these include Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. When isolating mRNA, methods known in the art can be used, for example, by using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo) or RNeasy Fibrous Tissue Mini Kit (Qiagen) according to the manufacturer's recommended protocol. In this way, the expression level of AAK1 can be measured.

[0151] The intracellular protein expression levels of HPCAL1, NDST3, CALN1, or AAK1 can be assayed using various methods known in the art. Specifically, these include, for example, immunoprecipitation, Western blot analysis (*immunoblot), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS).

[0152] In the evaluation or screening of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters, when screening or evaluating HPCAL1 gene inhibitors using inhibition of HPCAL1 function as an indicator, when screening or evaluating NDST3 gene inhibitors using inhibition of NDST3 function as an indicator, when screening or evaluating CALN1 gene inhibitors using inhibition of CALN1 function as an indicator, or when screening or evaluating AAK1 gene promoters using promotion of AAK1 function as an indicator, for example, the phosphorylation inhibitory activity of tau protein or the suppression of abnormal localization of TDP-43 protein in the cytoplasm can be used as an indicator in cells differentiated into neurons from iPS cells derived from MAPT mutant cells to which bafilomycin was added as a stress inducer or from cells derived from healthy individuals.

[0153] Furthermore, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters can also be evaluated or screened using animal models of neurological diseases. For example, a transgenic mouse overexpressing the MAPT gene can be used as a neurological disease model. The overexpressed MAPT gene may contain mutations. Another example of a neurological disease model mouse is a transgenic mouse overexpressing the TARDBP (TDP-43) gene. The overexpressed TARDBP gene may contain mutations.

[0154] For example, these MAPT-overexpressing transgenic mice show increased tau protein phosphorylation compared to normal mice that do not overexpress the MAPT gene. Therefore, by administering HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters, tau protein phosphorylation is inhibited, and this inhibition of tau protein phosphorylation can be used as an indicator to evaluate or screen HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters.

[0155] Furthermore, these TARDBP-overexpressing transgenic mice exhibit increased abnormal localization of TDP-43 protein in the cytoplasm compared to normal mice that do not overexpress the TARDBP gene. Therefore, administering an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, or an AAK1 gene promoter suppresses the abnormal localization of TDP-43 protein in the cytoplasm. This abnormal localization of TDP-43 protein in the cytoplasm can then be used as an indicator to evaluate or screen for HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters.

[0156] Furthermore, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters can be evaluated or screened using improvement of pathological conditions such as nerve fiber breakdown and cell death in animal models of neurodegenerative diseases as an indicator.

[0157] The HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters obtained by the selection method of the present invention can be used as preventive and / or therapeutic agents for neurodegenerative diseases, particularly tauopathy, and for the prevention and / or treatment of TDP-43-related diseases, in order to suppress the onset or progression of neurodegenerative diseases. In other words, the present invention provides a screening method for preventive and / or therapeutic agents for neurodegenerative diseases, comprising selecting at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters by the method described above. The screening method preferably includes evaluating both the HPCAL1 gene inhibitory ability, NDST3 gene inhibitory ability, CALN1 gene inhibitory ability, or AAK1 gene promoting ability of the candidate substance, and the HPCAL1 gene inhibitory ability, NDST3 gene inhibitory ability, CALN1 gene inhibitory ability, or AAK1 gene promoting ability, such as inhibition of phosphorylation of tau protein in nerve cells or suppression of abnormal localization of TDP-43 protein in the cytoplasm.

[0158] <Treatment of neurodegenerative diseases with HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters> In the present invention, neurodegenerative diseases can be treated or prevented by at least one substance selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters.

[0159] HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters may be used individually or in combination. Combinations of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters include: combination of HPCAL1 gene inhibitor and NDST3 gene inhibitor, combination of HPCAL1 gene inhibitor and CALN1 gene inhibitor, combination of HPCAL1 gene inhibitor and AAK1 gene promoter, combination of NDST3 gene inhibitor and CALN1 gene inhibitor, combination of NDST3 gene inhibitor and AAK1 gene promoter, CA Examples include combinations of an LN1 gene inhibitor and an AAK1 gene promoter, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene promoter, combinations of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter, and combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter.

[0160] Among neurodegenerative diseases, tauopathy, a neurodegenerative disease characterized by the abnormal accumulation of tau protein within cells and subsequent nerve loss, and TDP-43-related diseases, a neurodegenerative disease characterized by the abnormal localization of TDP-43 protein in the cytoplasm and subsequent nerve loss, are preferred.

[0161] The aforementioned tauopathy includes primary tauopathy and secondary tauopathy. Primary tauopathy is tauopathy in which the accumulation of tau protein is the direct cause, and examples include progressive supranuclear palsy (PSP), tauopathy-associated frontotemporal lobar degeneration (FTLD-tau), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), Pick's disease, frontotemporal dementia, globular glial tauopathy, argyrophilic grain disease, primary age-related tauopathy, and aging-related tau astrogliopathy.

[0162] The aforementioned secondary tauopathy refers to tauopathy caused by accumulation of other substances in addition to tau protein accumulation, and examples include Alzheimer's disease, chronic traumatic encephalopathy (CTE), multiple system atrophy (MSA), Guam Parkinsonism-dementia complex, and Guadeloupean parkinsonism.

[0163] HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters inhibit the phosphorylation of tau protein by inhibiting the function or expression of HPCAL1, NDST3, CALN1, and AAK1, respectively. This suppresses the accumulation of phosphorylated tau protein in nerve cells and inhibits the aggregation of phosphorylated tau protein.

[0164] Therefore, it is effective in treating and preventing the neurodegenerative diseases described above, which are associated with the aggregation of phosphorylated tau protein in nerve cells.

[0165] In particular, PSP is a disease in which tau protein abnormally aggregates in nerve cells and glial cells in the brain, leading to neuronal cell death. The HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, and AAK1 gene promoter of the present invention inhibit the phosphorylation of tau protein in the cytoplasm and inhibit the aggregation of phosphorylated tau protein, thereby suppressing neuronal cell death, maintaining and restoring nerve cells, and exhibiting therapeutic and / or preventive effects against PSP.

[0166] Examples of TDP-43-related diseases include amyotrophic lateral sclerosis (ALS), TDP-associated frontotemporal lobar degeneration (FTLD-TDP), limbic-dominant senile TDP-43 encephalopathy (LATE), and Perry syndrome.

[0167] HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters inhibit the function or expression of HPCAL1, NDST3, CALN1, and AAK1, respectively, thereby suppressing the translocation of TDP-43 protein from the nucleus to the cytoplasm, or promoting the degradation of cytoplasmic TDP-43, and suppressing the abnormal localization of TDP-43 protein in the cytoplasm.

[0168] Therefore, it is effective in treating and preventing neurodegenerative diseases such as TDP-43-related diseases, in which abnormal localization of the TDP-43 protein occurs in the cytoplasm and nerve loss occurs.

[0169] In other words, the agent of the present invention may be an agent that suppresses neurodegeneration or an agent that maintains or restores the function of nerve cells.

[0170] Accordingly, the present invention relates to a therapeutic or prophylactic agent for neurodegenerative diseases or a pharmaceutical composition for the treatment or prevention of neurodegenerative diseases comprising at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter as an active ingredient; a method for the treatment or prevention of neurodegenerative diseases comprising administering an effective amount of at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter to a subject requiring treatment or prevention of neurodegenerative diseases; and HPCAL1 for the treatment or prevention of neurodegenerative diseases. The present invention provides a method for preventing or treating neurodegenerative diseases, comprising: at least one selected from the group consisting of gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters; the use of at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters in the manufacture of pharmaceuticals for the treatment or prevention of neurodegenerative diseases; and at least one means selected from the group consisting of means for inhibiting the HPCAL1 gene, means for inhibiting the NDST3 gene, means for inhibiting the CALN1 gene, and means for promoting the AAK1 gene.

[0171] The active ingredients, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, may be used individually or in combination. Combinations of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters include: combination of HPCAL1 gene inhibitor and NDST3 gene inhibitor, combination of HPCAL1 gene inhibitor and CALN1 gene inhibitor, combination of HPCAL1 gene inhibitor and AAK1 gene promoter, combination of NDST3 gene inhibitor and CALN1 gene inhibitor, combination of NDST3 gene inhibitor and AAK1 gene promoter, CA Examples include combinations of an LN1 gene inhibitor and an AAK1 gene promoter, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene promoter, combinations of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter, and combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter.

[0172] The means for inhibiting the HPCAL1 gene, the means for inhibiting the NDST3 gene, the means for inhibiting the CALN1 gene, and the means for promoting the AAK1 gene may be used individually or in combination. Combinations of the means for inhibiting the HPCAL1 gene, the means for inhibiting the NDST3 gene, the means for inhibiting the CALN1 gene, and the means for promoting the AAK1 gene include: a combination of the means for inhibiting the HPCAL1 gene and the means for inhibiting the NDST3 gene; a combination of the means for inhibiting the HPCAL1 gene and the means for inhibiting the CALN1 gene; a combination of the means for inhibiting the HPCAL1 gene and the means for promoting the AAK1 gene; a combination of the means for inhibiting the NDST3 gene and the means for inhibiting the CALN1 gene; a combination of the means for inhibiting the NDST3 gene and the means for promoting the AAK1 gene; and CALN Examples include combinations of means to inhibit one gene and means to promote the AAK1 gene, combinations of means to inhibit the HPCAL1 gene, means to inhibit the NDST3 gene and means to inhibit the CALN1 gene, combinations of means to inhibit the HPCAL1 gene, means to inhibit the NDST3 gene and means to promote the AAK1 gene, combinations of means to inhibit the NDST3 gene, means to inhibit the CALN1 gene and means to promote the AAK1 gene, and combinations of means to inhibit the HPCAL1 gene, means to inhibit the NDST3 gene and means to inhibit the CALN1 gene and means to promote the AAK1 gene.

[0173] The agent of the present invention can be prepared by combining at least one substance selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, or a pharmaceutically acceptable salt thereof, either directly or with a pharmacologically acceptable carrier. As the pharmacologically acceptable carrier, various conventional organic or inorganic carrier substances can be used as formulation materials, and are incorporated as excipients, lubricants, binders, or disintegrants in solid formulations; or as solvents, solubilizers, suspending agents, isotonic agents, buffers, or analgesics in liquid formulations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, and sweeteners may be used as needed.

[0174] Furthermore, if the HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter is a nucleic acid, the agent of the present invention may include a nucleic acid delivery reagent.

[0175] The agent of the present invention can be administered orally or parenterally to subjects requiring treatment or prevention of neurodegenerative diseases. Parenteral administration methods include, for example, subcutaneous, intravenous, intramuscular, intra-arterial, and intraperitoneal administration. Administration may be continuous or long-term, or short-term or intermittent.

[0176] For oral administration, possible dosage forms include tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules and microcapsules), syrups, emulsions, and suspensions.

[0177] On the other hand, for parenteral administration, possible dosage forms include injections, infusions, drip infusions, and suppositories. Combining the drug with an appropriate base to create a sustained-release formulation is also effective.

[0178] As a method for formulating HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters into the above-mentioned dosage forms, known manufacturing methods commonly used in the field can be applied. Furthermore, when preparing the above-mentioned dosage forms, appropriate amounts of various pharmaceutical additives such as excipients, binders, disintegrants, lubricants, sweeteners, surfactants, suspending agents, and emulsifiers, which are commonly used in the pharmaceutical field when preparing such dosage forms, may be appropriately included in the manufacturing process.

[0179] For example, for parenteral administration, injectable preparations are preferably used. Injectable preparations include intravenous injections, as well as subcutaneous injections, intradermal injections, intramuscular injections, and intravenous drip injections. Such injectable preparations are prepared by dissolving, suspending, or emulsifying, for example, an HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter, such as the above-mentioned viral vector, in a sterile aqueous or oily solution commonly used for injectable preparations, according to a method known to the extent that the preparation is prepared.

[0180] The proportion of the active ingredient contained in the agent of the present invention, namely the HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter, can be appropriately set within a range that can produce the desired effect, but is usually 0.01 to 100% by weight, preferably 0.1 to 99.9% by weight, and more preferably 0.5 to 99.5% by weight.

[0181] The agent of the present invention, which contains at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters as an active ingredient, is stable, low-toxicity, and safe to use. The daily dose is not determined by the type of active ingredient, the weight and age of the recipient, symptoms, etc., but can be selected in the range of 0.1 ng to 1000 mg per kg of body weight per dose. Furthermore, when the active ingredient is a viral vector, for example, 1 × 10⁶ per kg of body weight of a human subject. ３ Vector genome (vg) / kg ~ 1 × 10⁻⁶ ３０ It can be expressed as vg / kg.

[0182] The number of times the agent of the present invention is administered is not particularly limited, but is usually about 1 to 30 times per month. If the symptoms of the disease recur after a reasonable interval, the agent of the present invention can be administered again.

[0183] The agents of the present invention are intended for use in animals requiring treatment or prevention of neurodegenerative diseases, including mammals such as mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cattle, horses, sheep, monkeys, and humans, with primates being preferred and humans being particularly preferred.

[0184] The present invention provides a method for preventing or treating neurodegenerative diseases, comprising at least one means selected from the group consisting of means for inhibiting the HPCAL1 gene, means for inhibiting the NDST3 gene, means for inhibiting the CALN1 gene, and means for promoting the AAK1 gene. Means for inhibiting the HPCAL1 gene include administering an HPCAL1 gene inhibitor to a subject in whom inhibition of the HPCAL1 gene is necessary. Means for inhibiting the NDST3 gene include administering an NDST3 gene inhibitor to a subject in whom inhibition of the NDST3 gene is necessary. Means for inhibiting the CALN1 gene include administering a CALN1 gene inhibitor to a subject in whom inhibition of the CALN1 gene is necessary. Means for promoting the AAK1 gene include administering an AAK1 gene promoter to a subject in whom promotion of the AAK1 gene is necessary. The administration of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, or AAK1 gene promoters can be carried out in the same manner as the administration of the agent of the present invention, and the subjects can be those targeted for administration as described above.

[0185] The following describes another embodiment of the present invention. In the following description, the main points to be described are those that differ from the embodiments described above, and for matters not specifically described, the matters described herein shall apply as appropriate. The matters and embodiments described herein may be applied one or more in combination.

[0186] <Tau Protein Phosphorylation Inhibitor and Method for Inhibiting Phosphorylation> In one embodiment, the present invention relates to a tau protein phosphorylation inhibitor. In another embodiment, the present invention relates to a method for inhibiting the phosphorylation of tau protein. The tau protein phosphorylation inhibitor contains as an active ingredient at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter. The method for inhibiting the phosphorylation of tau protein includes administering an effective amount of at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter to a subject in need.

[0187] The active ingredients, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, may be used individually or in combination. Combinations of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters include: combination of HPCAL1 gene inhibitor and NDST3 gene inhibitor, combination of HPCAL1 gene inhibitor and CALN1 gene inhibitor, combination of HPCAL1 gene inhibitor and AAK1 gene promoter, combination of NDST3 gene inhibitor and CALN1 gene inhibitor, combination of NDST3 gene inhibitor and AAK1 gene promoter, CA Examples include combinations of an LN1 gene inhibitor and an AAK1 gene promoter, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene promoter, combinations of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter, and combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter. The HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, AAK1 gene promoter and tau protein are as described above.

[0188] To determine whether tau protein phosphorylation is inhibited or suppressed, methods such as ELISA, Western blotting, flow cytometry, and immunohistochemistry can be used to determine the increase or decrease in the amount of phosphorylated tau protein in response to exposure to at least one substance selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters. The phosphorylated tau protein used for this determination is preferably the phosphorylated tau protein found in PSP patients, in which the Ser202 and Thr205 residues are phosphorylated. Furthermore, this determination is preferably performed using a system in which bafilomycin is added to nerve cells as a stress inducer to promote tau protein phosphorylation. Bafilomycin can promote tau protein phosphorylation by inhibiting lysosomal acidification in cells, thereby suppressing tau protein degradation and promoting tau protein accumulation.

[0189] <An inhibitor of abnormal localization of TDP-43 protein in the cytoplasm and a method for inhibiting abnormal localization of TDP-43 protein in the cytoplasm> In one embodiment, the present invention relates to an inhibitor of abnormal localization of TDP-43 protein in the cytoplasm. In another embodiment, the present invention relates to a method for inhibiting abnormal localization of TDP-43 protein in the cytoplasm. The inhibitor of abnormal localization of TDP-43 protein in the cytoplasm comprises, as an active ingredient, at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter. The method for inhibiting abnormal localization of TDP-43 protein in the cytoplasm comprises administering an effective amount of at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter to a subject requiring it.

[0190] The active ingredients, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, may be used individually or in combination. Combinations of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters include: combination of HPCAL1 gene inhibitor and NDST3 gene inhibitor, combination of HPCAL1 gene inhibitor and CALN1 gene inhibitor, combination of HPCAL1 gene inhibitor and AAK1 gene promoter, combination of NDST3 gene inhibitor and CALN1 gene inhibitor, combination of NDST3 gene inhibitor and AAK1 gene promoter, CA Examples include combinations of an LN1 gene inhibitor and an AAK1 gene promoter, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene promoter, combinations of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter, and combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter. The HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, AAK1 gene promoter and tau protein are as described above.

[0191] Suppressing the abnormal localization of TDP-43 protein in the cytoplasm means that the ratio of TDP-43 protein expression levels in the cytoplasm and nucleus, when the test substance (HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter) is brought into contact with various cells such as neurons, is lower than when the step of bringing the test substance (HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, or AAK1 gene promoter) into contact with the cells is not included, and is 90% or less, preferably 70% or less, and more preferably 50% or less.

[0192] Methods for measuring the abnormal localization of TDP-43 protein in the cytoplasm include the steps of: contacting a test substance with cells expressing TDP-43 protein; measuring the nuclear and / or cytoplasmic expression levels of TDP-43 protein in the cells that have been contacted with the test substance; and comparing the measured nuclear and cytoplasmic expression levels of TDP-43 protein, and / or the ratio of cytoplasmic to nuclear TDP-43 protein expression levels (calculated by dividing the cytoplasmic expression level of TDP-43 by the nuclear expression level) with the nuclear and cytoplasmic expression levels of TDP-43 protein and / or the ratio of cytoplasmic to nuclear TDP-43 protein expression levels in cells that have not been contacted with the test substance.

[0193] The method for bringing the test substance into contact with cells expressing the TDP-43 protein is not particularly limited. For example, the test substance can be administered to various cells such as neurons for exposure, and this method is applicable in vitro, in vivo, and ex vivo. Examples of cells include motor neurons differentiated from iPS cells derived from patients with TDP-43-related diseases, preferably motor neurons differentiated from iPS cells derived from ALS patients, and more preferably motor neurons differentiated from iPS cells derived from ALS patients having a TDP-43 gene mutation. These cells may also be subjected to a step in which a stress-inducing agent such as tunicamycin is administered. The expression level of TDP-43 protein in the nucleus and / or cytoplasm can be measured by conventional methods, such as immunofluorescence staining or Western blotting of nuclear and / or cytoplasmic proteins extracted from cells.

[0194] <Cytoplasmic TDP-43 Protein Degradation Inhibitor and Method for Inhibiting Cytoplasmic TDP-43 Degradation> In one embodiment, the present invention relates to a cytoplasmic TDP-43 protein degradation inhibitor. In another embodiment, the present invention relates to a method for inhibiting the degradation of cytoplasmic TDP-43 protein. The cytoplasmic TDP-43 protein degradation inhibitor contains as an active ingredient at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter. The method for inhibiting the degradation of cytoplasmic TDP-43 protein includes administering an effective amount of at least one selected from the group consisting of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene promoter to a subject requiring it.

[0195] The active ingredients, HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, may be used individually or in combination. Combinations of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters include: combination of HPCAL1 gene inhibitor and NDST3 gene inhibitor, combination of HPCAL1 gene inhibitor and CALN1 gene inhibitor, combination of HPCAL1 gene inhibitor and AAK1 gene promoter, combination of NDST3 gene inhibitor and CALN1 gene inhibitor, combination of NDST3 gene inhibitor and AAK1 gene promoter, CA Examples include combinations of an LN1 gene inhibitor and an AAK1 gene promoter, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor, combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene promoter, combinations of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter, and combinations of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene promoter. The HPCAL1 gene inhibitor, NDST3 gene inhibitor, CALN1 gene inhibitor, AAK1 gene promoter and TDP-43 protein are as described above.

[0196] Suppressing the degradation of cytoplasmic TDP-43 protein means that when the process includes contacting the test substance, an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, or an AAK1 gene promoter, with various cells such as neurons, the amount of cytoplasmic TDP-43 protein increases compared to when the process does not include contacting the test substance, an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, or an AAK1 gene promoter, with the cells, and the increase is 1.3 times or more, preferably 1.5 times or more, and more preferably 2.0 times or more.

[0197] Methods for measuring the inhibition of cytoplasmic TDP-43 protein degradation include the steps of: contacting a test substance with cells expressing TDP-43 protein; measuring the amount of cytoplasmic TDP-43 protein in the cells that have been contacted with the test substance; measuring the amount of cytoplasmic TDP-43 protein in cells that have not been contacted with the test substance; and comparing the amount of cytoplasmic TDP-43 protein in the cells that have been contacted with the test substance with the amount of cytoplasmic TDP-43 protein in cells that have not been contacted with the test substance.

[0198] The method for bringing the test substance into contact with cells expressing the TDP-43 protein is not particularly limited. For example, the test substance can be administered to various cells such as neurons for exposure, and this method is applicable in vitro, in vivo, and ex vivo. Examples of cells include motor neurons differentiated from iPS cells derived from patients with TDP-43-related diseases, preferably motor neurons differentiated from iPS cells derived from ALS patients, and more preferably motor neurons differentiated from iPS cells derived from ALS patients having a TDP-43 gene mutation. These cells may also be subjected to a step in which a stress-inducing agent such as tunicamycin is administered. The amount of cytoplasmic TDP-43 protein can be measured by conventional methods, such as immunofluorescence staining or Western blotting of cytoplasmic proteins extracted from cells.

[0199] With respect to the embodiments described above, this specification further discloses the following embodiments: <1> A preventive or therapeutic agent for neurodegenerative diseases containing an HPCAL1 gene inhibitor and an NDST3 gene inhibitor as active ingredients. <2> A preventive or therapeutic agent for neurodegenerative diseases containing an HPCAL1 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <3> A preventive or therapeutic agent for neurodegenerative diseases containing an HPCAL1 gene inhibitor and an AAK1 gene promoter as active ingredients. <4> A preventive or therapeutic agent for neurodegenerative diseases containing an NDST3 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <5> A preventive or therapeutic agent for neurodegenerative diseases containing an NDST3 gene inhibitor and an AAK1 gene promoter as active ingredients. <6> A preventive or therapeutic agent for neurodegenerative diseases containing a CALN1 gene inhibitor and an AAK1 gene promoter as active ingredients. <7> A preventive or therapeutic agent for neurodegenerative diseases, comprising an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and a CALN1 gene inhibitor as active ingredients. <8> A preventive or therapeutic agent for neurodegenerative diseases, comprising an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <9> A preventive or therapeutic agent for neurodegenerative diseases, comprising an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <10> A preventive or therapeutic agent for neurodegenerative diseases, comprising an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <11> A preventive or therapeutic agent according to any one of <1> to <10>, wherein the neurodegenerative disease is tauopathy. <12> The preventive or therapeutic agent according to any one of <11>, wherein the tauopathy is a primary tauopathy. <13> The preventive or therapeutic agent according to <12>, wherein the primary tauopathy is selected from the group consisting of progressive supranuclear palsy, tauopathy-associated frontotemporal lobar degeneration, corticobasal degeneration, argyrophilic grain dementia, and Pick's disease. <14> The preventive or therapeutic agent according to <11>, wherein the tauopathy is a secondary tauopathy.<15> The preventive or therapeutic agent according to <14>, wherein the secondary tauopathy is selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, and multiple system atrophy. <16> The preventive or therapeutic agent according to any one of <1> to <15>, wherein the prevention or treatment of the neurodegenerative disease is by inhibition of phosphorylation of tau protein. <17> The preventive or therapeutic agent according to any one of <1> to <10>, wherein the neurodegenerative disease is a TDP-43 related disease. <18> The preventive or therapeutic agent according to <17>, wherein the TDP-43 related disease is selected from the group consisting of amyotrophic lateral sclerosis, TDP-associated frontotemporal lobar degeneration, limbic system-dominant senile TDP-43 encephalopathy, and Perry syndrome. <19> The preventive or therapeutic agent according to <17> or <18>, wherein the prevention or treatment of the neurodegenerative disease is by suppressing the abnormal localization of the TDP-43 protein in the cytoplasm. <20> The preventive or therapeutic agent according to any one of <1> to <3>, <7>, <8> and <10>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <21> The preventive or therapeutic agent according to <20>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene. <22> The preventive or therapeutic agent according to <20>, wherein the antibody is an antibody against the HPCAL1 gene product. <23> The preventive or therapeutic agent according to any one of <1>, <4>, <5> and <7> to <10>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <24> The preventive or therapeutic agent according to <23>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <25> The preventive or therapeutic agent according to <23>, wherein the antibody is an antibody against the NDST3 gene product.<26> The preventive or therapeutic agent according to any one of <2>, <4>, <6>, <7>, <9>, and <10>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <27> The preventive or therapeutic agent according to <26>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene. <28> The preventive or therapeutic agent according to <26>, wherein the antibody is an antibody against the CALN1 gene product. <29> The preventive or therapeutic agent according to any one of <3>, <5>, <6>, and <8> to <10>, wherein the AAK1 gene promoting substance is a substance containing nucleic acid. <30> The preventive or therapeutic agent according to <29>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an AAK1 gene enhancing nucleic acid and an AAK1 gene expression vector. <31> The preventive or therapeutic agent according to <30>, wherein the AAK1 gene expression vector is a viral vector. <32> The preventive or therapeutic agent according to <30> or <31>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <33> The preventive or therapeutic agent according to <32>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <34> A tau protein phosphorylation inhibitor containing an HPCAL1 gene inhibitor and an NDST3 gene inhibitor as active ingredients. <35> A tau protein phosphorylation inhibitor containing an HPCAL1 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <36> A tau protein phosphorylation inhibitor containing an HPCAL1 gene inhibitor and an AAK1 gene promoter as active ingredients. <37> A tau protein phosphorylation inhibitor containing an NDST3 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <38> A tau protein phosphorylation inhibitor containing an NDST3 gene inhibitor and an AAK1 gene promoter as active ingredients.<39> A tau protein phosphorylation inhibitor containing a CALN1 gene inhibitor and an AAK1 gene promoter as active ingredients. <40> A tau protein phosphorylation inhibitor containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and a CALN1 gene inhibitor as active ingredients. <41> A tau protein phosphorylation inhibitor containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <42> A tau protein phosphorylation inhibitor containing an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <43> A tau protein phosphorylation inhibitor containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <44> The inhibitor according to <34> to <36>, <40>, <41> and <43>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <45> The inhibitor according to <44>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene. <46> The inhibitor according to <44>, wherein the antibody is an antibody against the HPCAL1 gene product. <47> The inhibitor according to any one of <34>, <37>, <38> and <40> to <43>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <48> The inhibitor according to <47>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <49> The inhibitor according to <47>, wherein the antibody is an antibody against the NDST3 gene product. <50> The inhibitor according to any one of <35>, <37>, <39>, <40>, <42>, and <43>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.<51> The inhibitor according to <50>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the CALN1 gene. <52> The inhibitor according to <50>, wherein the antibody is an antibody against the CALN1 gene product. <53> The inhibitor according to any one of <36>, <38>, <39>, and <41> to <43>, wherein the AAK1 gene promoting substance is a nucleic acid-containing substance. <54> The inhibitor according to <53>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of AAK1 gene enhancing nucleic acid and AAK1 gene expression vectors. <55> The inhibitor according to <54>, wherein the AAK1 gene expression vector is a viral vector. <56> The inhibitor according to <54> or <55>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <57> The inhibitor according to <56>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <58> An inhibitor of abnormal localization of TDP-43 protein to the cytoplasm, comprising an HPCAL1 gene inhibitor and an NDST3 gene inhibitor as active ingredients. <59> An inhibitor of abnormal localization of TDP-43 protein to the cytoplasm, comprising an HPCAL1 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <60> An inhibitor of abnormal localization of TDP-43 protein to the cytoplasm, comprising an HPCAL1 gene inhibitor and an AAK1 gene promoter as active ingredients. <61> An inhibitor of abnormal localization of TDP-43 protein to the cytoplasm, comprising an NDST3 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <62> An agent that suppresses the abnormal localization of TDP-43 protein into the cytoplasm, containing an NDST3 gene inhibitor and an AAK1 gene promoter as active ingredients. <63> An agent that suppresses the abnormal localization of TDP-43 protein into the cytoplasm, containing a CALN1 gene inhibitor and an AAK1 gene promoter as active ingredients.<64> An agent that inhibits the abnormal localization of TDP-43 protein into the cytoplasm, containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and a CALN1 gene inhibitor as active ingredients. <65> An agent that inhibits the abnormal localization of TDP-43 protein into the cytoplasm, containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <66> An agent that inhibits the abnormal localization of TDP-43 protein into the cytoplasm, containing an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <67> An agent that inhibits the abnormal localization of TDP-43 protein into the cytoplasm, containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <68> The inhibitor according to any one of <58> to <60>, <64>, <65> and <67>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <69> The inhibitor according to <68>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene. <70> The inhibitor according to <68>, wherein the antibody is an antibody against the HPCAL1 gene product. <71> The inhibitor according to any one of <58>, <61>, <62> and <64> to <67>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <72> The inhibitor according to <71>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <73> The inhibitor according to <71>, wherein the antibody is an antibody against the NDST3 gene product. <74> The inhibitor according to any one of <59>, <61>, <63>, <65>, <66>, and <67>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody.<75> The inhibitor according to <74>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the CALN1 gene. <76> The inhibitor according to <74>, wherein the antibody is an antibody against the CALN1 gene product. <77> The inhibitor according to any one of <60>, <62>, <63>, and <65> to <67>, wherein the AAK1 gene promoting substance is a nucleic acid-containing substance. <78> The inhibitor according to <77>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of AAK1 gene enhancing nucleic acid and AAK1 gene expression vectors. <79> The preventive or therapeutic agent according to <78>, wherein the AAK1 gene expression vector is a viral vector. <80> The inhibitor according to <78> or <79>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <81> The inhibitor according to <80>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <82> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an HPCAL1 gene inhibitor and an NDST3 gene inhibitor to a subject in need. <83> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an HPCAL1 gene inhibitor and a CALN1 gene inhibitor to a subject in need. <84> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an HPCAL1 gene inhibitor and an AAK1 gene promoter to a subject in need. <85> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an NDST3 gene inhibitor and a CALN1 gene inhibitor to a subject in need. <86> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an NDST3 gene inhibitor and an AAK1 gene promoter to a subject in need thereof.<87> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of a CALN1 gene inhibitor and an AAK1 gene promoter to a subject in need. <88> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and a CALN1 gene inhibitor to a subject in need. <89> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor and an AAK1 gene inhibitor to a subject in need. <90> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an NDST3 gene inhibitor, a CALN1 gene inhibitor and an AAK1 gene inhibitor to a subject in need. <91> A method for preventing or treating a neurodegenerative disease, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor to a subject in need thereof. <92> The method according to any one of <82> to <91>, wherein the neurodegenerative disease is a tauopathy. <93> The method according to any one of <92>, wherein the tauopathy is a primary tauopathy. <94> The method according to <93>, wherein the primary tauopathy is selected from the group consisting of progressive supranuclear palsy, tauopathy-associated frontotemporal lobar degeneration, corticobasal degeneration, argyrophilic grain dementia, and Pick's disease. <95> The method according to <92>, wherein the tauopathy is a secondary tauopathy. <96> The method according to <95>, wherein the secondary tauopathy is selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, and multiple system atrophy. <97> The preventive or therapeutic agent according to any one of <82> to <96>, wherein the prevention or treatment of the neurodegenerative disease is by inhibition of phosphorylation of tau protein. <98> The method according to any one of <82> to <91>, wherein the neurodegenerative disease is a TDP-43 related disease. <99> The method according to <98>, wherein the TDP-43 related disease is selected from the group consisting of amyotrophic lateral sclerosis, TDP-associated frontotemporal lobar degeneration, limbic system-dominant senile TDP-43 encephalopathy, and Perry syndrome.<100> The method according to <98> or <99>, wherein the prevention or treatment of the neurodegenerative disease is by suppressing the abnormal localization of the TDP-43 protein in the cytoplasm. <101> The method according to any one of <82> to <84>, <88>, <89> and <91>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <102> The method according to <101>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene. <103> The method according to <101>, wherein the antibody is an antibody against the HPCAL1 gene product. <104> The method according to any one of <82>, <85>, <86> and <88> to <91>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <105> The method according to <104>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <106> The method according to <104>, wherein the antibody is an antibody against the NDST3 gene product. <107> The method according to any one of <83>, <85>, <87>, <88>, <90>, and <91>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <108> The method according to <107>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene. <109> The method according to <107>, wherein the antibody is an antibody against the CALN1 gene product. <110> The method according to any one of <84>, <86>, <87> and <89> to <91>, wherein the AAK1 gene promoting substance is a substance containing nucleic acid.<111> The method according to <110>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector. <112> The method according to <111>, wherein the AAK1 gene expression vector is a viral vector. <113> The method according to <111> or <112>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <114> The method according to <113>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <115> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an HPCAL1 gene inhibitor and an NDST3 gene inhibitor to a subject requiring the same. <116> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an HPCAL1 gene inhibitor and a CALN1 gene inhibitor to a subject requiring the same. <117> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an HPCAL1 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <118> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an NDST3 gene inhibitor and a CALN1 gene inhibitor to a subject requiring the same. <119> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an NDST3 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <120> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of a CALN1 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <121> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and a CALN1 gene inhibitor to a subject requiring the same. <122> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and an AAK1 gene inhibitor to a subject requiring such inhibitors.<123> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor to a subject requiring the same. <124> A method for inhibiting the phosphorylation of tau protein, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor to a subject requiring the same. <125> The method according to any one of <115> to <117>, <121>, <122>, and <124>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <126> The method according to <125>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide against the HPCAL1 gene, siRNA, shRNA, and a vector expressing at least one of these. <127> The method according to <125>, wherein the antibody is an antibody against the HPCAL1 gene product. <128> The method according to any one of <115>, <118>, <119> and <121> to <124>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <129> The method according to <128>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide against the NDST3 gene, siRNA, shRNA, and a vector expressing at least one of these. <130> The method according to <128>, wherein the antibody is an antibody against the NDST3 gene product. <131> The method according to any one of <116>, <118>, <120>, <121>, <123> and <124>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <132> The method according to <131>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene. <133> The method according to <131>, wherein the antibody is an antibody against the CALN1 gene product.<134> The method according to any one of <117>, <119>, <120> and <122> to <124>, wherein the AAK1 gene promoting substance is a substance containing nucleic acid. <135> The method according to <134>, wherein the substance containing nucleic acid includes at least one selected from the group consisting of AAK1 gene enhancing nucleic acid and AAK1 gene expression vector. <136> The method according to <135>, wherein the AAK1 gene expression vector is a viral vector. <137> The method according to <135> or <136>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <138> The method according to <137>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <139> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of an HPCAL1 gene inhibitor and an NDST3 gene inhibitor to a subject requiring the same. <140> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of an HPCAL1 gene inhibitor and a CALN1 gene inhibitor to a subject requiring the same. <141> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of an HPCAL1 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <142> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of an NDST3 gene inhibitor and a CALN1 gene inhibitor to a subject requiring the same. <143> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of an NDST3 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <144> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of a CALN1 gene inhibitor and an AAK1 gene promoter to a subject requiring the same.<145> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of HPCAL1 gene inhibitor, NDST3 gene inhibitor, and CALN1 gene inhibitor to a subject requiring the use of these substances. <146> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of HPCAL1 gene inhibitor, NDST3 gene inhibitor, and AAK1 gene inhibitor to a subject requiring the use of these substances. <147> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of NDST3 gene inhibitor, CALN1 gene inhibitor, and AAK1 gene inhibitor to a subject requiring the use of these substances. <148> A method for suppressing abnormal localization of TDP-43 protein in the cytoplasm, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor to a subject requiring such inhibitor. <149> The method according to any one of <139> to <141>, <145>, <146>, and <148>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <150> The method according to <149>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the HPCAL1 gene. <151> The method according to <149>, wherein the antibody is an antibody against the HPCAL1 gene product. <152> The method according to any one of <139>, <142>, <143> and <145> to <148>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <153> The method according to <152>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <154> The method according to <152>, wherein the antibody is an antibody against the NDST3 gene product.<155> The method according to any one of <140>, <142>, <144>, <145>, <147> and <148>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <156> The method according to <155>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene. <157> The method according to <155>, wherein the antibody is an antibody against the CALN1 gene product. <158> The method according to any one of <141>, <143>, <144> and <146> to <148>, wherein the AAK1 gene promoter is a nucleic acid-containing substance. <159> The method according to <158>, wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector. <160> The method according to <159>, wherein the AAK1 gene expression vector is a viral vector. <161> The method according to <159> or <160>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <162> The method according to <161>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <163> A cytoplasmic TDP-43 protein degradation inhibitor containing an HPCAL1 gene inhibitor and an NDST3 gene inhibitor as active ingredients. <164> A cytoplasmic TDP-43 protein degradation inhibitor containing an HPCAL1 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <165> A cytoplasmic TDP-43 protein degradation inhibitor containing an HPCAL1 gene inhibitor and an AAK1 gene promoter as active ingredients. <166> A cytoplasmic TDP-43 protein degradation inhibitor containing an NDST3 gene inhibitor and a CALN1 gene inhibitor as active ingredients. <167> A cytoplasmic TDP-43 protein degradation inhibitor containing an NDST3 gene inhibitor and an AAK1 gene promoter as active ingredients.<168> A cytoplasmic TDP-43 protein degradation inhibitor containing a CALN1 gene inhibitor and an AAK1 gene promoter as active ingredients. <169> A cytoplasmic TDP-43 protein degradation inhibitor containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and a CALN1 gene inhibitor as active ingredients. <170> A cytoplasmic TDP-43 protein degradation inhibitor containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <171> A cytoplasmic TDP-43 protein degradation inhibitor containing an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <172> A cytoplasmic TDP-43 protein degradation inhibitor containing an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <173> The inhibitor according to <163> to <165>, <169>, <170> and <172>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <174> The inhibitor according to <173>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide against the HPCAL1 gene, siRNA, shRNA, and a vector expressing at least one of these. <175> The inhibitor according to <173>, wherein the antibody is an antibody against the HPCAL1 gene product. <176> The inhibitor according to any one of <163>, <166>, <167> and <169> to <172>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <177> The inhibitor according to <176>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <178> The inhibitor according to <176>, wherein the antibody is an antibody against the NDST3 gene product.<179> The inhibitor according to any one of <164>, <166>, <168>, <169>, <171>, and <172>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <180> The inhibitor according to <179>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene. <181> The inhibitor according to <179>, wherein the antibody is an antibody against the CALN1 gene product. <182> The inhibitor according to any one of <165>, <167>, <168>, and <170> to <172>, wherein the AAK1 gene promoter is a substance containing nucleic acid. <183> The inhibitor according to <182>, wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector. <184> The inhibitor according to <183>, wherein the AAK1 gene expression vector is a viral vector. <185> The inhibitor according to <183> or <184>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <186> The inhibitor according to <185>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors. <187> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an HPCAL1 gene inhibitor and an NDST3 gene inhibitor to a subject requiring the same. <188> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an HPCAL1 gene inhibitor and a CALN1 gene inhibitor to a subject requiring the same. <189> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an HPCAL1 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <190> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an NDST3 gene inhibitor and a CALN1 gene inhibitor to a subject requiring the same.<191> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an NDST3 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <192> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of a CALN1 gene inhibitor and an AAK1 gene promoter to a subject requiring the same. <193> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and a CALN1 gene inhibitor to a subject requiring the same. <194> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, and an AAK1 gene inhibitor to a subject requiring the same. <195> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising administering an effective amount of an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor to a subject requiring the same. <196> A method for inhibiting the degradation of cytoplasmic TDP-43 protein, comprising an HPCAL1 gene inhibitor, an NDST3 gene inhibitor, a CALN1 gene inhibitor, and an AAK1 gene inhibitor as active ingredients. <197> The method according to <187> to <189>, <193>, <194>, and <196>, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody. <198> The method according to <197>, wherein the substance containing nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide against the HPCAL1 gene, siRNA, shRNA, and a vector expressing at least one of these. <199> The method according to <197>, wherein the antibody is an antibody against the HPCAL1 gene product. <200> The method according to any one of <187>, <190>, <191> and <193> to <196>, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.<201> The method according to <200>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene. <202> The method according to <200>, wherein the antibody is an antibody against the NDST3 gene product. <203> The method according to any one of <188>, <190>, <192>, <193>, <195>, and <196>, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a nucleic acid-containing substance and an antibody. <204> The method according to <203>, wherein the nucleic acid-containing substance comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene. <205> The method according to <203>, wherein the antibody is an antibody against the CALN1 gene product. <206> The method according to any one of <189>, <191>, <192> and <194> to <196>, wherein the AAK1 gene promoting substance is a substance containing nucleic acid. <207> The method according to <206>, wherein the substance containing nucleic acid includes at least one selected from the group consisting of AAK1 gene enhancing nucleic acid and AAK1 gene expression vector. <208> The method according to <207>, wherein the AAK1 gene expression vector is a viral vector. <209> The method according to <207> or <208>, wherein the AAK1 gene expression vector is selected from the group consisting of DNA-containing viral vectors, retroviral vectors, and lentiviral vectors. <210> The method according to <209>, wherein the DNA-containing viral vector is selected from the group consisting of adenovirus vectors and adeno-associated virus vectors.

[0200] Certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, but the following examples are merely illustrative of the compounds described herein and are not intended to limit them. Each of the references cited herein is incorporated herein by reference in its entirety.

[0201] The present invention will be described in detail below with reference to examples, but unless otherwise specified, the present invention is not limited to the following.

[0202] The present disclosure will be further described below with reference to examples. However, the scope of the present disclosure is not limited to these examples. Unless otherwise specified, "%v / v" means "volume / volume%". In each figure, the group represented by "**" indicates that P < 0.01 is statistically significant. In each figure, the group represented by "*" indicates that P < 0.05 is statistically significant.

[0203] Example 1 Taking PSP as an example of a type of tauopathy, a disease model for evaluating the accumulation of phosphorylated tau protein and cytotoxicity was constructed using the following method.

[0204] First, human induced plurippotent stem (iPS) cells were induced from cells derived from human PSP patients and cells derived from healthy individuals using standard methods. For the cells derived from PSP patients, cells with PSP-risk mutations in the MAPT gene (MAPT mutant cells) were used. Next, each iPS cell was differentiated into excitatory neurons (hereinafter, excitatory neurons will also be simply referred to as "neurons") using standard methods.

[0205] To preserve cells for long-term storage, neurons were sometimes frozen 2 to 7 days after differentiation, as needed. Nerves that have undergone this freezing process are also called "frozen neurons."

[0206] Next, neurons were cultured using a 48-well culture plate (Corning, 3548) under the conditions shown below.

[0207] The culture plates were coated in advance before cell seeding. Specifically, a 0.02% Poly-L-Ornithine solution (Sigma-Aldrich, P4957) was added to each well of the culture plate, and the plates were incubated at 37°C and 5% CO2. ２After standing in an incubator for 2 hours, each well was washed and incubated with 20 μg / mL Laminin solution (Thermo Fisher Scientific, 23017015) at 37°C and 5% CO2. ２ The process was carried out by allowing the cells to stand in an incubator for an additional two hours. Subsequently, the frozen neurons were thawed using a conventional method, and the thawed neurons were seeded at a rate of 50,000 cells / well in each well and cultured in the presence of a culture medium having the following composition.

[0208] The culture medium used was a mixture with the following composition.

[0209] ・DMEM / F12 (manufactured by Thermo Fisher Scientific, 21331-020): 50% v / v ・Neurobasal Medium (manufactured by Thermo Fisher Scientific, 21103-049): 50% v / v ・Glutamax Supplement (manufactured by Thermo Fisher Scientific, 35050061): 1% v / v ・Penicillin-Streptomycin (10000 units / mL) (Thermo Fisher Scientific, 15140-148): 0.5% v / v ・Component N (manufactured by Elixirgen): 3% v / v ・Component G2 (manufactured by Elixirgen): 0.1% v / v ・Component P (manufactured by Elixirgen): 0.05% v / v

[0210] The culture medium containing a solution of the stress-inducing agent Dimethyl sulfoxide (DMSO) (final concentration: bafilomycin (BA) 1-5 nM, DMSO 0.03% v / v) was applied to neurons after 7 days of culture, and the occurrence of cell damage was evaluated over time. Separately, a group without the stress-inducing agent (a group with the same concentration of DMSO added) was also prepared. Cell damage was evaluated using live cell imaging with IncuCyte S3 (Sartorius) according to the attached protocol, and the neurite length per unit area (unit: mm / mm) was measured. ２The neurite length was analyzed over time and used as an evaluation index. A smaller neurite length value indicates that cell damage has occurred.

[0211] Cell Damage Assessment (1) Figures 1A and 1B show the results of the neurite length in each experimental group, calculated as a ratio based on the results at the start of stress-inducing agent treatment (0hr). Figure 1A shows the results using neurons derived from healthy individuals, and Figure 1B shows the results using MAPT-mutated neurons. A smaller value on the vertical axis indicates that the neurite length decreases over time, and that cell damage is occurring. The experimental groups in Figures 1A and 1B are as follows: ・Healthy-DMSO: A group using neurons derived from healthy individuals, without a stress-inducing agent. ・Healthy-bafilomycin: A group using neurons derived from healthy individuals, with a stress-inducing agent. ・MAPT-DMSO: A group using MAPT-mutated neurons, without a stress-inducing agent. ・MAPT-bafilomycin: A group using MAPT-mutated neurons, with a stress-inducing agent.

[0212] When bafilomycin treatment was applied to neurons derived from healthy individuals and neurons with the MAPT mutation, a decrease in neurite length was observed in the bafilomycin-treated group, and this decrease was more pronounced in MAPT-mutated neurons than in neurons derived from healthy individuals.

[0213] Evaluation of cytoplasmic accumulation of phosphorylated tau protein (1) Cytoplasmic accumulation of tau protein with phosphorylation at Ser202 and Thr205 residues, observed in PSP patients, was evaluated by immunofluorescence staining of neurons. Cellular neurons were washed with Phosphate Buffered Saline (PBS) and fixed with 4% Paraformaldehyde (PFA). After blocking with PBS containing 5% Fetal Bovine Serum (FBS) and 0.1% Triton-X, a primary antibody dilution containing anti-phosphorylated tau protein antibody and anti-β-III tubulin antibody was added and incubated overnight at 4°C. The anti-β-III tubulin antibody was used to visualize the cell bodies of neurons. The following day, after washing with PBS, a secondary antibody dilution conjugated with Alexa dye was added and incubated at room temperature for 1 hour. After further nuclear staining, the cells were washed with PBS and imaged using a fluorescence microscope. The captured images were analyzed using Matlab (Mathworks), and the fluorescence intensity of phosphorylated tau protein in the cytoplasmic region was calculated.

[0214] The results of cytoplasmic accumulation of phosphorylated tau protein are shown in Figures 2A and 2B. Figure 2A shows the image obtained by fluorescence microscopy, and Figure 2B shows the fluorescence intensity of phosphorylated tau protein in the cytoplasmic region. The experimental groups in Figures 2A and 2B are as follows: • Healthy-DMSO: A group using neurons derived from healthy individuals, without a stress-inducing agent. • Healthy-bafilomycin: A group using neurons derived from healthy individuals, with a stress-inducing agent. • MAPT-DMSO: A group using MAPT-mutated neurons, without a stress-inducing agent. • MAPT-bafilomycin: A group using MAPT-mutated neurons, with a stress-inducing agent.

[0215] In Figures 2A and 2B, higher fluorescence intensity values ​​indicate an accumulation of phosphorylated tau protein in the cytoplasm, reproducing a phenomenon similar to that observed in brain tissue from PSP patients.

[0216] Example 2 Using the experimental model described above, the effect of an HPCAL1 gene inhibitor on neuronal cell damage was evaluated. The HPCAL1 gene inhibitor is an siRNA (siHPCAL1) containing the sequence represented by SEQ ID NO: 32 (nucleotide sequence: GAAGAAUUCAUCAGAGGUG) as the sense strand and SEQ ID NO: 33 (nucleotide sequence: CACCUCUGAUGAAUUCUUC) as the antisense strand, designed for the nucleic acid sequence of human HPCAL1. This HPCAL1 gene inhibitor is configured to inhibit the production of human HPCAL1 transcripts and / or translation products in cells, thereby reducing the amount of HPCAL1 protein in cells. Separately, a control siRNA (siNeg) that does not target the nucleic acid sequence of human HPCAL1 was prepared. This siNeg does not fall under the category of an HPCAL1 gene inhibitor as defined herein.

[0217] In the experimental model described above, on day 4 of culture, siNeg or siHPCAL1 was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. RNA was collected on day 7 of culture, and the knockdown efficiency was measured by quantitative PCR. The HPCAL1 expression in the siNeg group in each neuron was set to 100%, and the results for each group were expressed as a percentage of that. As shown in Figure 3, HPCAL1 gene expression was suppressed in the group exposed to the HPCAL1 gene inhibitor.

[0218] In the experimental model described above, on day 4 of culture, HPCAL1 siRNA was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. Then, on day 7 of culture, a stress inducer (or DMSO alone) was added to the final concentration mentioned above, and the cells were cultured for a maximum of 48 hours in the presence of the stress inducer.

[0219] Cell Damage Evaluation (2) Using MAPT mutant neurons, the occurrence of neuronal cell damage was evaluated by morphological analysis of cells in the same manner as in Example 1. The results are shown in Figure 4. The experimental groups shown in Figure 4 are as follows: ・siNeg-bafilomycin: A group containing a stress inducer but not containing an HPCAL1 gene inhibitor ・siHPCAL1-bafilomycin: A group containing both a stress inducer and an HPCAL1 gene inhibitor The degree of cell damage was calculated based on neurite length, and the degree of cell damage in the siNeg-bafilomycin group in each neuron was set to 100%, with the results for each group expressed as a percentage. As shown in Figure 4, the occurrence or progression of cell damage was significantly suppressed in the group exposed to the HPCAL1 gene inhibitor.

[0220] Evaluation of Phosphorylated Tau Protein Accumulation (2) Using MAPT mutant neurons, the accumulation of phosphorylated tau protein was measured in the same manner as in Example 1, with and without the HPCAL1 gene inhibitor. The results are shown in Figure 5. The experimental groups shown in Figure 5 are as follows. In Figure 5, the value for the siNeg-DMSO group in each neuron is set to 100%, and the results for each group are expressed as a percentage of that. ・siNeg-bafilomycin: Group containing a stress inducer and not containing the HPCAL1 gene inhibitor ・siHPCAL1-bafilomycin: Group containing a stress inducer and the HPCAL1 gene inhibitor

[0221] As shown in Figure 5, inhibiting the production of HPCAL1 protein as a translation product reduced the accumulation of phosphorylated tau protein in MAPT mutant neurons. Therefore, by using an HPCAL1 gene inhibitor, the accumulation of cytoplasmic phosphorylated tau protein can be suppressed, improving the ability of cells such as excitatory neurons to perform their inherent functions.

[0222] Example 3 Using the experimental model described above, the effect of an NDST3 gene inhibitor on neuronal cell damage was evaluated. The NDST3 gene inhibitor is a siRNA (siNDST3) containing the sequence represented by Sequence ID No. 34 (nucleotide sequence: GCUGGCACGUGGAUGAUUA) as the sense strand and Sequence ID No. 35 (nucleotide sequence: UAAUCAUCCACGUGCCAGC) as the antisense strand, designed for the nucleic acid sequence of human NDST3. This NDST3 gene inhibitor is configured to inhibit the production of human NDST3 transcripts and / or translation products in cells, thereby reducing the amount of NDST3 protein in cells. Separately, a control siRNA (siNeg) that does not target the nucleic acid sequence of human NDST3 was created. This siNeg does not fall under the category of an NDST3 gene inhibitor as defined herein.

[0223] In the experimental model described above, on day 4 of culture, siNeg or siNDST3 was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. RNA was collected on day 7 of culture, and the knockdown efficiency was measured by quantitative PCR. The NDST3 expression in the siNeg group in each neuron was set to 100%, and the results for each group were expressed as a percentage of that. As shown in Figure 6, NDST3 gene expression was suppressed in the group exposed to the NDST3 gene inhibitor.

[0224] In the experimental model described above, on day 4 of culture, NDST3 siRNA was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. Then, on day 7 of culture, a stress inducer (or DMSO alone) was added to the final concentration mentioned above, and the cells were cultured for a maximum of 48 hours in the presence of the stress inducer.

[0225] Cell Damage Evaluation (2) Using MAPT mutant neurons, the occurrence of neuronal cell damage was evaluated by morphological analysis of cells in the same manner as in Example 1. The results are shown in Figure 7. The experimental groups shown in Figure 7 are as follows: ・siNeg-bafilomycin: A group containing a stress inducer and not containing an NDST3 gene inhibitor ・siNDST3-bafilomycin: A group containing both a stress inducer and an NDST3 gene inhibitor The degree of cell damage was calculated based on neurite length, and the degree of cell damage in the siNeg-bafilomycin group in each neuron was set to 100%, with the results for each group expressed as a percentage. As shown in Figure 7, the occurrence or progression of cell damage was significantly suppressed in the group exposed to the NDST3 gene inhibitor.

[0226] Evaluation of Phosphorylated Tau Protein Accumulation (2) Using MAPT mutant neurons, the accumulation of phosphorylated tau protein was measured in the same manner as in Example 1, with and without the NDST3 gene inhibitor. The results are shown in Figure 8. The experimental groups shown in Figure 8 are as follows. In Figure 8, the value for the siNeg-DMSO group in each neuron is set to 100%, and the results for each group are expressed as a percentage of that. ・siNeg-bafilomycin: Group containing a stress inducer and not containing the NDST3 gene inhibitor ・siNDST3-bafilomycin: Group containing a stress inducer and the NDST3 gene inhibitor

[0227] As shown in Figure 8, inhibiting the production of NDST3 protein as a translation product reduced the accumulation of phosphorylated tau protein in MAPT mutant neurons. Therefore, by using an NDST3 gene inhibitor, the accumulation of cytoplasmic phosphorylated tau protein can be suppressed, improving the ability of cells such as excitatory neurons to perform their inherent functions.

[0228] Example 4 Using the experimental model described above, the effect of a CALN1 gene inhibitor on neuronal cell damage was evaluated. The CALN1 gene inhibitor is an siRNA (siCALN1) containing the sequence represented by SEQ ID NO: 36 (nucleotide sequence: GCGAACAGCUGGCUAAUAU) as the sense strand and SEQ ID NO: 37 (nucleotide sequence: AUAUUAGCCAGCUGUUCGC) as the antisense strand, designed for the nucleic acid sequence of human CALN1. This CALN1 gene inhibitor is configured to inhibit the production of human CALN1 transcripts and / or translation products in cells, thereby reducing the amount of CALN1 protein in cells. Separately, a control siRNA (siNeg) that does not target the nucleic acid sequence of human CALN1 was prepared. This siNeg does not fall under the category of a CALN1 gene inhibitor as defined herein.

[0229] In the experimental model described above, on day 4 of culture, siNeg or siCALN1 was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. RNA was collected on day 7 of culture, and the knockdown efficiency was measured by quantitative PCR. The results for each group are expressed as a percentage of the CALN1 expression in the siNeg group in each neuron, with CALN1 expression set to 100%. As shown in Figure 9, CALN1 gene expression was suppressed in the group containing the CALN1 gene inhibitor.

[0230] In the experimental model described above, on day 4 of culture, CALN1 siRNA was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. Then, on day 7 of culture, a stress inducer (or DMSO alone) was added to the final concentration mentioned above, and the cells were cultured for a maximum of 48 hours in the presence of the stress inducer.

[0231] Cell Damage Evaluation (2) Using MAPT mutant neurons, the occurrence of neuronal cell damage was evaluated by morphological analysis of cells in the same manner as in Example 1. The results are shown in Figure 10. The experimental groups shown in Figure 10 are as follows: ・siNeg-bafilomycin: A group containing a stress inducer and not containing a CALN1 gene inhibitor ・siCALN1-bafilomycin: A group containing a stress inducer and a CALN1 gene inhibitor The degree of cell damage was calculated based on neurite length, and the degree of cell damage in the siNeg-bafilomycin group in each neuron was set to 100%, with the results for each group expressed as a percentage. As shown in Figure 10, the occurrence or progression of cell damage was significantly suppressed in the group exposed to the CALN1 gene inhibitor.

[0232] Evaluation of Phosphorylated Tau Protein Accumulation (2) Using MAPT mutant neurons, the accumulation of phosphorylated tau protein was measured in the same manner as in Example 1, with and without the CALN1 gene inhibitor. The results are shown in Figure 11. The experimental groups shown in Figure 11 are as follows. In Figure 11, the value for the siNeg-DMSO group in each neuron is set to 100%, and the results for each group are expressed as a percentage of that. ・siNeg-bafilomycin: Group containing a stress inducer and not containing the CALN1 gene inhibitor ・siCALN1-bafilomycin: Group containing a stress inducer and the CALN1 gene inhibitor

[0233] As shown in Figure 11, inhibiting the production of CALN1 protein as a translation product reduced the accumulation of phosphorylated tau protein in MAPT mutant neurons. Therefore, by using a CALN1 gene inhibitor, the accumulation of cytoplasmic phosphorylated tau protein can be suppressed, improving the ability of cells such as excitatory neurons to perform their inherent functions.

[0234] Example 5 Using the experimental model described above, the effect of an AAK1 gene promoter on neuronal cell damage was evaluated. The AAK1 gene promoter was a lentiviral vector containing the nucleic acid sequence represented by SEQ ID NO: 23, which encodes the wild-type human AAK1 protein consisting of the amino acid sequence represented by SEQ ID NO: 28. This vector was prepared by a conventional method. This promoter is configured to promote the production of human AAK1 transcripts and / or translation products in cells, thereby increasing the amount of AAK1 protein in the cells. Separately, a lentiviral vector (Null) that does not contain the nucleic acid encoding the wild-type AAK1 protein was prepared. This lentiviral vector does not correspond to the AAK1 gene promoter as defined herein.

[0235] In the experimental model described above, on day 3 of culture, each of the aforementioned lentiviral vectors was added to the culture plate of each neuron to achieve a predetermined multiplicity of infection (MOI) (e.g., MOI 1), and the culture was continued. Then, on day 7 of culture, RNA was collected, and the rate of expression increase was measured by quantitative PCR. The AAK1 expression in the Null group in each neuron was set to 100%, and the results for each group were expressed as a percentage of that. As shown in Figure 12, the AAK1 gene expression level increased in the group exposed to the AAK1 gene-promoting substance.

[0236] In the experimental model described above, on day 3 of culture, each of the aforementioned lentiviral vectors was added to the culture plate of each neuron to achieve a predetermined multiplicity of infection (MOI) (e.g., MOI 1), and the culture was continued. Then, on day 7 of culture, a stress inducer (or DMSO alone) was added to the final concentration described above, and the cells were cultured for a maximum of 48 hours in the presence of the stress inducer.

[0237] Cell Damage Evaluation (2) Using MAPT mutant neurons, the occurrence of neuronal cell damage was evaluated by morphological analysis of cells in the same manner as in Example 1. The results are shown in Figure 13. The experimental groups shown in Figure 13 are as follows: ・Null-bafilomycin: A group containing a stress inducer and not containing the AAK1 gene promoter ・AAK1-bafilomycin: A group containing both a stress inducer and the AAK1 gene promoter The degree of cell damage was calculated based on neurite length, and the degree of cell damage in the Null-bafilomycin group in each neuron was set to 100%, with the results for each group expressed as a percentage. As shown in Figure 13, the occurrence or progression of cell damage was significantly suppressed in the group exposed to the AAK1 gene promoter.

[0238] Evaluation of Phosphorylated Tau Protein Accumulation (2) Using MAPT mutant neurons, the accumulation of phosphorylated tau protein was measured in the same manner as in Example 1, with and without the promoting substance. The results are shown in Figure 14. The experimental groups shown in Figure 14 are as follows. In Figure 14, the value for the Null-DMSO group in each neuron is set to 100%, and the results for each group are expressed as a percentage of that. Null-bafilomycin: Group containing a stress inducer and not containing an AAK1 gene promoting substance AAK1-bafilomycin: Group containing a stress inducer and an AAK1 gene promoting substance

[0239] As shown in Figure 14, promoting the production of AAK1 protein as a translation product reduced the accumulation of phosphorylated tau protein in MAPT mutant neurons. Therefore, by using an AAK1 gene promoter, the accumulation of cytoplasmic phosphorylated tau protein can be suppressed, improving the ability of cells such as excitatory neurons to perform their inherent functions.

[0240] Example 6 Using ALS as an example of a TDP-43-related disease, a disease model was constructed to evaluate cell damage, and HPCAL1 expression inhibitors were evaluated. First, human induced plurippotent stem (iPS) cells were induced from cells derived from human ALS patients and cells derived from healthy individuals using conventional methods. Cells derived from ALS patients were those known to have heterozygous mutations in the TARDBP gene that increase the risk of developing ALS. Next, motor neurons (hereinafter, motor neurons are also simply referred to as "neurons") were differentiated from each iPS cell using conventional methods. For long-term preservation of the cells, the neurons were frozen 2 to 7 days after differentiation, as needed. Neurons that have undergone the freezing process are also referred to as "frozen neurons."

[0241] Next, neurons were cultured in a 96-well culture plate (Corning, 3599) under the conditions described below. The culture plate was coated in advance before cell seeding. Specifically, 0.02% Poly-L-Ornithine solution (Sigma-Aldrich, P4957) was added to each well of the culture plate, and the cells were cultured at 37°C under 5% CO2. ２ After standing in an incubator for 2 hours, each well was washed and incubated with 20 μg / mL Laminin solution (Thermo Fisher Scientific, 23017015) at 37°C and 5% CO2. ２ The process was carried out by allowing the cells to stand in an incubator for an additional two hours. Subsequently, the frozen neurons were thawed using a conventional method, and the thawed neurons were seeded at a rate of 25,000 cells / well in each well and cultured in the presence of a culture medium having the following composition.

[0242] The culture medium used was a mixture with the following composition.・DMEM / F12 (manufactured by Thermo Fisher Scientific, 21331-020): 50% v / v ・Neurobasal Medium (manufactured by Thermo Fisher Scientific, 21103-049): 50% v / v ・Glutamax Supplement (manufactured by Thermo Fisher Scientific, 35050061): 1% v / v ・Penicillin-Streptomycin (10000 units / mL) (Thermo Fisher Scientific, 15140-148): 0.5% v / v ・Component N1 (manufactured by Elixirgen): 3% v / v ・Component A (manufactured by Elixirgen): 0.1% v / v ・Component D4 (manufactured by Elixirgen): 0.1% v / v ・Component P (manufactured by Elixirgen): 0.05% v / v

[0243] To evaluate the cytoplasmic localization of TDP-43, on day 4 of culture, siNeg or siHPCAL1, used in Example 2, was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. Subsequently, the culture medium, in which a stress-inducing agent dimethyl sulfoxide (DMSO) solution (final concentration: tunicamycin 0.1 μg / mL, DMSO 0.01% v / v) was dissolved, was brought into contact with the neurons after 7 days of culture. Separately, a group without the stress-inducing agent (a group with the same concentration of DMSO added) was also prepared.

[0244] The cytoplasmic localization of the TDP-43 protein was evaluated by immunofluorescence staining of neurons. Cultured neurons were washed with Phosphate Buffered Saline (PBS) and fixed with 4% Paraformaldehyde (PFA). After blocking with PBS containing 5% Fetal Bovine Serum (FBS) and 0.1% Triton-X, a primary antibody dilution containing anti-TDP-43 antibody and anti-β-III tubulin antibody was added, and the cells were incubated overnight at 4°C. The anti-β-III tubulin antibody was used to visualize the neuronal cell bodies. The following day, after washing with PBS, a secondary antibody dilution conjugated with Alexa dye was added, and the cells were incubated at room temperature for 1 hour. After further nuclear staining, the cells were washed with PBS and imaged using a fluorescence microscope. The captured images were analyzed using Matlab (Mathworks), and the fluorescence intensity of the TDP-43 protein in the nuclear and cytoplasmic regions was calculated, respectively.

[0245] Figure 15 shows the results of the localization of TDP-43 protein into the cytoplasm. The experimental groups in Figure 15 are as follows: • siNeg-DMSO: A group using neurons derived from ALS patients, without stress inducers or HPCAL1 gene inhibitors. • siNeg-tunicamycin: A group using neurons derived from ALS patients, containing stress inducers but without HPCAL1 gene inhibitors. • siHPCAL1-tunicamycin: A group using neurons derived from ALS patients, containing stress inducers and HPCAL1 gene inhibitors.

[0246] The cytoplasmic localization of TDP-43 protein shown in Figure 15 is expressed as the ratio of the TDP-43 fluorescence intensity in the cytoplasm to that in the nucleus (in Figure 15, "cytoplasm / nucleus ratio"). A higher ratio indicates a higher proportion of TDP-43 protein localization in the cytoplasm, suggesting localization abnormalities. The results for each group are expressed as a percentage, with the value for the siNeg-DMSO group in each neuron set to 100%.

[0247] As shown in Figure 15, inhibiting the production of HPCAL1 protein as a translation product resulted in a decrease in the cytoplasm / nucleus ratio in the siHPCAL1-tunicamycin group compared to the siNeg-tunicamycin group. Therefore, by using an HPCAL1 gene inhibitor, it is possible to improve the abnormal localization and clearance of TDP-43 protein in the cytoplasm, thereby improving the ability of cells such as motor neurons to perform their inherent functions.

[0248] Example 7 Similar to Example 6, ALS was used as an example of a TDP-43-related disease, and cell damage induced by an NDST3 expression inhibitor was evaluated. Each iPS cell was differentiated into motor neurons using the same method as in Example 6. Subsequently, neurons were cultured in a 96-well culture plate (Corning, 3599) under the conditions shown below. The culture plate was coated in advance before cell seeding. Specifically, 0.02% Poly-L-Ornithineyoueki (Sigma-Aldrich, P4957) was added to each well of the culture plate, and the cells were cultured at 37°C in 5% CO2. ２ After standing in an incubator for 2 hours, each well was washed and incubated with 20 μg / mL Laminin solution (Thermo Fisher Scientific, 23017015) at 37°C in 5% CO2. ２ The process was carried out by allowing the cells to stand in an incubator for an additional two hours. Subsequently, the frozen neurons were thawed using a conventional method, and the thawed neurons were seeded at a rate of 25,000 cells / well in each well and cultured in the presence of a culture medium having the following composition.

[0249] The culture medium used was a mixture with the following composition.・DMEM / F12 (manufactured by Thermo Fisher Scientific, 21331-020): 50% v / v ・Neurobasal Medium (manufactured by Thermo Fisher Scientific, 21103-049): 50% v / v ・Glutamax Supplement (manufactured by Thermo Fisher Scientific, 35050061): 1% v / v ・Penicillin-Streptomycin (10000 units / mL) (Thermo Fisher Scientific, 15140-148): 0.5% v / v ・Component N1 (manufactured by Elixirgen): 3% v / v ・Component A (manufactured by Elixirgen): 0.1% v / v ・Component D4 (manufactured by Elixirgen): 0.1% v / v ・Component P (manufactured by Elixirgen): 0.05% v / v

[0250] On day 4 of the cytotoxicity evaluation culture, siNeg or siNDST3 used in Example 3 was added to each neuron's culture plate to a predetermined concentration (10 nM), and the culture was continued. Subsequently, the culture medium, in which a stress-inducing agent dimethyl sulfoxide (DMSO) solution (final concentration: tunicamycin 0.1 μg / mL, DMSO 0.01% v / v) was dissolved, was brought into contact with the neurons on day 7 of the culture.

[0251] Figure 16 shows the results of evaluating the occurrence of neuronal cell damage by morphological analysis of cells. The experimental groups in Figure 16 are as follows: • siNeg-tunicamycin: A group using neurons derived from ALS patients, containing a stress inducer but not containing an NDST3 gene inhibitor. • siNDST3-tunicamycin: A group using neurons derived from ALS patients, containing a stress inducer and an NDST3 gene inhibitor.

[0252] As shown in Figure 16, inhibiting the production of NDST3 protein as a translation product suppressed the occurrence or progression of cell damage in the siNDST3-tunicamycin group compared to the siNeg-tunicamycin group. Therefore, by using an NDST3 gene inhibitor, it is possible to improve the function of cells such as motor neurons so that they can perform their inherent functions.

Claims

1. A preventive or therapeutic agent for neurodegenerative diseases, comprising at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters, as an active ingredient.

2. The preventive or therapeutic agent according to claim 1, wherein the neurodegenerative disease is tauopathy.

3. The preventive or therapeutic agent according to claim 2, wherein the tauopathy is a primary tauopathy.

4. The preventive or therapeutic agent according to claim 3, wherein the primary tauopathy is selected from the group consisting of progressive supranuclear palsy, tauopathy-associated frontotemporal lobar degeneration, corticobasal degeneration, argyrophilic granule dementia, and Pick's disease.

5. The preventive or therapeutic agent according to claim 2, wherein the tauopathy is a secondary tauopathy.

6. The preventive or therapeutic agent according to claim 5, wherein the secondary tauopathy is selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, and multiple system atrophy.

7. The preventive or therapeutic agent according to claim 1 or 2, wherein the prevention or treatment of the neurodegenerative disease is by inhibition of tau protein phosphorylation.

8. The preventive or therapeutic agent according to claim 1, wherein the neurodegenerative disease is a TDP-43 related disease.

9. The preventive or therapeutic agent according to claim 8, wherein the TDP-43 related disease is selected from the group consisting of amyotrophic lateral sclerosis, TDP-associated frontotemporal lobar degeneration, limbic system-dominant senile TDP-43 encephalopathy, and Perry syndrome.

10. The preventive or therapeutic agent according to claim 1 or 2, wherein the HPCAL1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

11. The preventive or therapeutic agent according to claim 10, wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and vectors expressing at least one of these against the HPCAL1 gene.

12. The preventive or therapeutic agent according to claim 1 or 2, wherein the NDST3 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

13. The preventive or therapeutic agent according to claim 12, wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA, and a vector expressing at least one of these against the NDST3 gene.

14. The preventive or therapeutic agent according to claim 1 or 2, wherein the CALN1 gene inhibitor comprises at least one selected from the group consisting of a substance containing nucleic acid and an antibody.

15. The preventive or therapeutic agent according to claim 14, wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of an antisense oligonucleotide, siRNA, shRNA, and a vector expressing at least one of these against the CALN1 gene.

16. The preventive or therapeutic agent according to claim 1 or 2, wherein the AAK1 gene promoting substance is a substance containing nucleic acid.

17. The preventive or therapeutic agent according to claim 16, wherein the substance containing the nucleic acid comprises at least one selected from the group consisting of AAK1 gene-enhancing nucleic acid and AAK1 gene expression vector.

18. The preventive or therapeutic agent according to claim 17, wherein the AAK1 gene expression vector is a viral vector.

19. A phosphorylation inhibitor of tau protein containing at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters as an active ingredient.

20. An agent for inhibiting the abnormal localization of TDP-43 protein in the cytoplasm, comprising at least one selected from the group consisting of HPCAL1 gene inhibitors, NDST3 gene inhibitors, CALN1 gene inhibitors, and AAK1 gene promoters as an active ingredient.

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