Prophylactic and / or therapeutic agent for neurodegenerative disease

Abstract

The purpose of the present invention is to provide a novel prophylactic and / or therapeutic agent for a neurodegenerative disease, which can increase the number of new neurons in an adult brain.　The present invention is a prophylactic and / or therapeutic agent for a neurodegenerative disease, the agent containing, as an active ingredient, an inhibitor of any one of genes shown in table 1 or a protein derived from any one of the genes.

Description

Preventive and / or therapeutic agent for neurodegenerative diseases

[0001] The present invention relates to a novel method for activating neurogenesis and suppressing amyloid-β deposition in the adult brain, and to a preventive and / or therapeutic agent for neurodegenerative diseases using this method.

[0002] It has become clear that neural stem cells and neural progenitor cells exist in the adult brain of mammals, including humans, although their absolute numbers are small, and that neurogenesis occurs in some brain regions. However, the majority of neural stem cells in the adult brain are quiescent and have low proliferation and neurogenesis capacities. This proliferation and neurogenesis capacity further declines with age, resulting in almost no new neurons being born in the aging brain. A significant decrease in newborn neurons is thought to be a cause of brain dysfunction such as dementia, as well as neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0003] Under these circumstances, attempts have been made to promote functional recovery by amplifying and differentiating the neural stem cells and neural progenitor cells present in small numbers in the adult brain using stimulation with growth factors and other methods (see Non-Patent Document 1). However, the effectiveness of these attempts has not been sufficient. Furthermore, active research has been conducted on cell transplantation therapy models using neural stem cells and neural progenitor cells derived from fetal brain, ES cells, and iPS cells. However, it is unclear whether cell transplantation therapy in the brain can reconstruct normal neural circuits, and there is a significant risk of side effects. Furthermore, although many attempts have been made to increase the number of newly generated neurons in the adult brain, the effectiveness of these attempts has been limited (see Non-Patent Documents 1-5).

[0004] On the other hand, we previously demonstrated that aged neural stem cells, which have already lost their proliferative and neurogenic potential, can be functionally rejuvenated by inducing the expression of the zinc finger transcription factor gene Plagl2 and inhibiting the activity of Dyrk1a, a gene associated with Down syndrome (a genetic disease known to accelerate aging). These rejuvenated neural stem cells continuously proliferate at levels similar to those observed in the immature hippocampus, producing new neurons and improving cognition. We also found that this treatment stimulated neurogenesis, reduced Aβ deposition, and improved cognition in 5XFAD mice, a mouse model of Alzheimer's disease (Non-Patent Document 6, Patent Document 1).

[0005] International Publication No. 2022 / 186089

[0006] Nagahara AH et al. (2009) Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease, Nat.Med.,vol.15(3),pp.331-337.Henriette van Praag et al.,Exercise enhances learning and hippocampal neurogenesis in aged mice,J Neurosci.,2005 Sep 21;25(38):8680-5Benedetta Artegiani et al.,Overexpression of cdk4 and cyclinD1 triggers greater expansion of neural stem cells in the adult mouse brain. J Exp Med.2011 May 9;208(5):937-48Benraiss, A.et al.(2013) Mobilization of endogenous progenitor cells regenerates functionally-integrated medium spiny striopallidal projection neurons and delays disease progression in a transgenic model of Huntington's disease. Cell Stem Cell 12, 787-799Berdugo-Vega, G.et al.(2020). Increasing neurogenesis refines hippocampal activity rejuvenating navigational learning strategies and contextual memory throughout life. Nat. Commun. 11, 135Kaise, T.et.al., (2022) Functional rejuvenation of aged neural stem cells by Plagl2 and anti-Dyrk1a activity. Genes Dev 36: 23-37.

[0007] The present invention aims to provide a novel method for activating neurogenesis and inhibiting amyloid-β (Aβ) deposition in the adult brain, as well as to provide a preventive and / or therapeutic agent for neurodegenerative diseases using this method.

[0008] We previously demonstrated that administration of a lentivirus (iPaD) with Plagl2 expression-inducing activity and anti-Dyrk1a activity in 5XFAD mice, a mouse model of Alzheimer's disease, efficiently stimulated neurogenesis in the adult brain, reduced amyloid-β (Aβ) deposition, and improved cognitive function. Based on these results, we further investigated downstream events of iPaD treatment in 5XFAD mice. Global RNA-seq and functional analysis revealed that iPaD treatment in 5XFAD mice significantly reduced the expression of specific genes and significantly enhanced the expression of specific genes (ligand-receptor combinations). Furthermore, among the specific genes whose expression was decreased by iPaD treatment, knockdown of Prkag2 (protein kinase AMP-activated non-catalytic subunit gamma 2), Maml2 (Mastermind2), Gfra2 (glial cell line-derived neurotrophic factor receptor alpha-2), and Rpl29 (ribosomal protein L29) efficiently stimulated neurogenesis in the adult brain and reduced amyloid-β (Aβ) deposition. Since neurogenesis activation in the adult brain may improve age-related cognitive impairment and neurodegenerative diseases, knockdown of Prkag2, Gfra2, Maml2, and / or Rpl29 is strongly suggested to be a promising therapeutic strategy for such neurodegenerative diseases, including Alzheimer's disease.

[0009] That is, the present invention relates to nucleic acid medicines and the like that target specific genes whose expression has been shown to decrease downstream of iPaD treatment and that enable the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease by regulating their expression.

[0010] [1] A preventive and / or therapeutic agent for a neurodegenerative disease, comprising as an active ingredient an inhibitor of any of the genes listed in Table 1 below or a protein derived from any of the genes.

[0011] [2] The agent for preventing and / or treating a neurodegenerative disease according to [1], wherein the inhibitor is: (A) a vector having a polynucleotide comprising a nucleic acid sequence of a miR-shRNA (microRNA adapted short hairpin RNA) for any of the genes listed in Table 1 above and a promoter sequence operably linked to the nucleic acid sequence; (B) an siRNA, shRNA, or antisense oligonucleotide for any of the genes listed in Table 1 above; (C) an antibody for a protein derived from any of the genes listed in Table 1 above; or (D) a small molecule compound having an inhibitory effect on the expression and / or function of any of the genes listed in Table 1 above or a protein derived therefrom. [3] The agent for preventing and / or treating a neurodegenerative disease according to [1] or [2], wherein the gene is a mammalian gene. [4] The agent for preventing and / or treating a neurodegenerative disease according to [2] or [3], wherein the vector is a lentivirus vector, adeno-associated virus vector, adenovirus vector, or plasmid vector. [5] The agent for preventing and / or treating a neurodegenerative disease according to any of [1] to [4], which is administered by lumbar puncture. [6] A preventive and / or therapeutic agent for a neurodegenerative disease, comprising as an active ingredient an agent for promoting the expression and / or function of any of the ligand-receptor gene sets listed in Table 2 below, or a protein derived from any of the gene sets.

[0012] [7] The agent for preventing and / or treating neurodegenerative diseases according to any one of [1] to [6], wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and dementia caused by frontotemporal lobar degeneration. [8] The agent for preventing and / or treating neurodegenerative diseases according to any one of [1] to [6], wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease and Parkinson's disease. [9] The agent for preventing and / or treating neurodegenerative diseases according to any one of [1] to [6], wherein the neurodegenerative disease is Alzheimer's disease.

[0013] According to the present invention, suppressing or inhibiting the expression of the specific genes or proteins derived from any of these genes can efficiently activate neurogenesis in the adult brain and reduce amyloid-β deposition. Such activation of neurogenesis and reduction of amyloid-β deposition in the adult brain are thought to lead to improvements in age-related cognitive dysfunction and neurodegenerative diseases. Therefore, knockdown of the specific genes, such as Prkag2, Gfra2, Maml2, and Rpl29, or proteins derived from any of these genes, is strongly suggested to be a promising therapeutic strategy for neurodegenerative diseases, including Alzheimer's disease.

[0014] Figure 1A shows the long-term activation of neurogenesis in the brains of Alzheimer's disease model mice by iPaD treatment. Control or iPaD lentivirus was injected into the dentate gyrus of the hippocampus of 5-month-old 5XFAD mice, and brain sections were examined immunohistochemically at subsequent stages (4, 8, and 12 weeks postinfection (wpi)). Quantitative data for MCM2+ cells are shown in the bottom panel, and MCM2 expression at 4 wpi is shown in the top panel. Lentivirus-infected NSCs and their progeny are mCherry+. iPaD treatment increased the number of MCM2+-activated NSCs compared to controls (N = 4). Figure 1B shows the same experiment as Figure 1A, with quantitative data for DCX+ cells shown in the bottom panel and DCX expression at 4 wpi shown in the top panel. Lentivirus-infected NSCs and their progeny are mCherry+. iPaD treatment increased the number of DCX+ newborn neuroblasts compared to controls. N = 4. Figure 2 shows the inhibitory effect of iPaD treatment on Aβ deposition in the brains of Alzheimer's disease model mice. Control or iPaD lentivirus was injected into the dentate gyrus of the hippocampus of 5-month-old 5XFAD mice, and brain sections were examined immunohistochemically at subsequent stages (4, 8, and 12 wpi). Quantification data for Aβ deposition are shown on the right. iPaD treatment reduced Aβ deposition. N = 4. Figure 3 shows genes upregulated or downregulated in the mouse hippocampus by iPaD treatment. Control or iPaD lentivirus was injected into the dentate gyrus of 5-month-old 5XFAD mice, and the hippocampi were isolated 4 weeks later (4 wpi) and subjected to RNA-seq analysis. In the hippocampus of 5XFAD mice, iPaD treatment significantly upregulated or downregulated many genes (p < 0.05). Figure 4A shows the results of gene ontology (GO) analysis of 592 genes whose expression was significantly increased by iPaD treatment in the hippocampus of 5XFAD mice, and Figure 4B shows the results of gene ontology (GO) analysis of 462 genes whose expression was significantly decreased by iPaD treatment in the hippocampus of 5XFAD mice.Figure 5 shows a knockdown strategy using the miR-E backbone shRNA system, which contains mCherry and three repeats of a nuclear localization signal (3NLS) under the control of the ubiquitous elongation factor promoter (pEFS). This knockdown system was introduced into the dentate gyrus of 5XFAD mice using lentivirus. Figure 6A compares neurogenesis in 5XFAD mice following knockdown of Maml2, Ptgds, Ell3, Prkag2, Rpl29, Gfra2, Itpk1, and Mical3. 5-6-month-old 5XFAD mice were injected with lentivirus containing knockdown or control genes, and the brains were examined 8 weeks later. Prkag2 knockdown was the most effective in activating neurogenesis. N = 4. Figure 6B shows quantitative data for DCX+ cells from the same experiment as Figure 6A. Prkag2 knockdown was the most effective in activating neurogenesis. N = 4. Figure 7A shows the reduction of Aβ deposition by knockdown of Maml2, Ptgds, Ell3, Prkag2, Rpl29, Gfra2, Itpk1, and Mical3 in 5XFAD mice. Five- to six-month-old 5XFAD mice were injected with lentiviruses containing either the knockdown or control (Ctr) genes, and the brains were examined 8 weeks later. Knockdown of Prkag2 and Gfra2 was the most effective in reducing Aβ deposition. N = 4. Figure 7B shows quantitative data on Aβ deposition from the same experiment as in Figure 7A. Knockdown of Prkag2 and Gfra2 was the most effective in reducing Aβ deposition. N = 4. Figure 8 shows histological staining images of lipid droplets (white dots of BODIPY) and activated AMPK (white dots of pAMPK) in 4-month-old (wild-type and 5XFAD) and 8-month-old (5XFAD) mice. Figure 9 shows the results of Western blot analysis of pAMPK levels in cultured neural stem cells collected two days after infection with Prkag2 knockdown lentivirus (Prkag2 shRNA) or control lentivirus (Control). Figure 10 shows the results of qPCR analysis of mRNA levels of each AMPK subunit in cell extracts obtained in the same manner as in Figure 9.Figure 11 shows the results of quantifying EdU uptake in cultured neural stem cells 2 days after infection with Prkag2 knockdown (Prkag2 shRNA), Maml2 knockdown (Maml2 shRNA) lentivirus, or control lentivirus (Control). Figure 12 shows a schematic diagram of the results of a search for secreted factor-receptor pairs involved in AMPK signaling among the genes activated by iPaD. The Adipoq-Adipor2, Il33-Il1rl1, and Il17a-Il17rc pairs were identified. Figure 13 shows the results of qPCR analysis of mRNA levels of secreted factors involved in AMPK signaling in cell extracts collected 2 days after infection with iPaD lentivirus (iPaD) or control lentivirus (Control).

[0015] The present invention will be described in detail below. In this specification, unless otherwise specified, molecular biological techniques can be performed by methods described in general experimental manuals known to those skilled in the art or methods similar thereto. Furthermore, unless otherwise specified, the terms used in this specification should be interpreted as having the meanings commonly used in the technical field.

[0016] We previously demonstrated that administration of a lentivirus (iPaD) capable of inducing Plagl2 expression and inhibiting Dyrk1a activity in 5XFAD mice, a mouse model of Alzheimer's disease, efficiently stimulated neurogenesis in the adult brain, reduced amyloid-β deposition, and improved cognitive function. Based on these results, we further investigated downstream events of iPaD treatment in 5XFAD mice. Global RNA-seq and functional analysis revealed that iPaD treatment of 5XFAD mice significantly reduced the expression of specific genes listed in Table 1 and significantly increased the expression of specific genes (ligand-receptor combinations) listed in Table 2. Among the specific genes whose expression was decreased by iPaD treatment, knockdown of Prkag2 (protein kinase AMP-activated non-catalytic subunit gamma 2), Maml2 (mastermind-like transcriptional coactivator 2), Gfra2 (glial cell line-derived neurotrophic factor receptor alpha-2), and Rpl29 (ribosomal protein L29) resulted in efficient activation of neurogenesis in the adult brain and reduced amyloid-β deposition. Since activation of neurogenesis in the adult brain may improve age-related cognitive dysfunction and neurodegenerative diseases, these findings strongly suggest that inhibitors of specific genes, such as Prkag2 and Maml2, or proteins derived from these genes, as well as promoters of the expression and / or function of specific ligand-receptor gene sets, as shown in Table 2, or proteins derived from these gene sets, may be promising therapeutic strategies for neurodegenerative diseases, including Alzheimer's disease.

[0017] <Preventive and / or therapeutic agent for neurodegenerative disease> A first embodiment of the preventive and / or therapeutic agent for neurodegenerative disease of the present invention is characterized in that it contains, as an active ingredient, an inhibitor of any of the genes listed in Table 1 above, or a protein derived from any of the genes. Furthermore, a second embodiment of the preventive and / or therapeutic agent for neurodegenerative disease of the present invention is characterized in that it contains, as an active ingredient, an agent that promotes the expression and / or function of any of the ligand-receptor gene sets listed in Table 2 above, or a protein derived from any of the gene sets. Each embodiment is described below.

[0018] [First embodiment] A first embodiment of the agent for preventing and / or treating neurodegenerative diseases of the present invention is characterized in that it contains, as an active ingredient, an inhibitor of any of the genes listed in Table 1 above or a protein derived from any of the genes.

[0019] The nucleic acid sequences of the genes in Table 1 can be known sequences as the coding region (CDS) of the mRNA sequence of each of the above genes, and for example, the transcript sequence of each gene registered in the gene database Ensembl can be used. The nucleic acid sequences of the genes in Table 1 in the present invention are identical to, or have 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more homology with, the transcript sequences of the genes registered in Ensembl, and the proteins encoded by these sequences have the activity corresponding to each gene registered in the Ensembl nucleotide sequence database or the like.

[0020] The genes listed in Table 1 above are derived from mammals such as humans, monkeys, pigs, horses, cows, rabbits, sheep, goats, cats, dogs, guinea pigs, and mice, and are preferably derived from humans, monkeys, or mice, with humans being more preferred.

[0021] The inhibitor is not particularly limited as long as it inhibits or suppresses part or all of the expression and / or function of any of the genes listed in Table 1 above or a protein derived from any of the genes, but preferred examples include the following:

[0022] (A) A vector having a polynucleotide comprising the nucleic acid sequence of miR-shRNA (microRNA adapted short hairpin RNA) for any of the genes listed in Table 1 above and a promoter sequence operably linked to the nucleic acid sequence. (B) An siRNA, shRNA, or antisense oligonucleotide for any of the genes listed in Table 1 above. (C) An antibody against a protein derived from any of the genes listed in Table 1 above. (D) A low molecular weight compound having an inhibitory effect on the expression and / or function of any of the genes listed in Table 1 above or a protein derived therefrom. Each of these is explained below.

[0023] (A) A vector containing a polynucleotide comprising the nucleic acid sequence of a miR-shRNA (microRNA-adapted short hairpin RNA) for any of the genes listed in Table 1 and a promoter sequence operably linked to the nucleic acid sequence. The miR-shRNA (microRNA-adapted short hairpin RNA) for any of the genes listed in Table 1 is a type of shRNA (hairpin-shaped RNA sequence used for gene silencing by RNA interference) that can suppress the expression of the gene. shRNA is an RNA that can be degraded by Dicer in vivo to generate siRNA. shRNA has a stem-loop structure comprising a double-stranded stem and a hairpin loop. The sequence of this hairpin loop portion is not particularly limited, but can be a sequence of 5 to 12 bases (Kawasaki H. et al., Nucleic Acid Res. (2003) 31: 700-707). miR-shRNA is an shRNA adapted to the miRNA pathway, expressed by RNA polymerase II, and acts by being cleaved by both Drosha and Dicer. Such miR-shRNAs are not particularly limited as long as they have a nucleic acid sequence that can suppress the expression of the above-mentioned genes, and can be designed and appropriately produced based on the gene sequences shown in Table 1 above using methods well known to those skilled in the art.

[0024] In this embodiment, a promoter sequence is operably linked to the nucleic acid sequence of the miR-shRNA. Here, the "operably linked promoter sequence" may be a promoter sequence that functions in neural cells such as neural stem cells. Examples of such promoter sequences include the Hes5 promoter, GFAP promoter, Sox2 promoter, Lfng promoter, U6 promoter, and H1 promoter, which function in neural stem cells.

[0025] As used herein, "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter and / or enhancer) and a polynucleotide sequence of interest, which may be direct or indirect (when there is another intervening polynucleotide sequence). In the present disclosure, a promoter sequence directs transcription of a nucleic acid sequence of a linked miR-shRNA (microRNA adapted short hairpin RNA) of interest.

[0026] As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid," "gene," or "nucleic acid molecule," and refers to a polymer of nucleotides. As used herein, the term "nucleotide sequence" is used interchangeably with "nucleic acid sequence" or "base sequence," and is represented as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T). For example, a "polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof" refers to a polynucleotide or a fragment thereof comprising the sequence represented by each of the deoxynucleotides A, G, C, and / or T of SEQ ID NO: 1.

[0027] In the present invention, the polynucleotide may further contain known sequences such as an enhancer sequence that assists in the transcription of mRNA, translation into protein, etc., a Kozak sequence, and an appropriate polyadenylation signal sequence.

[0028] In the present invention, the configuration for introducing a polynucleotide into a target cell such as a neural stem cell is not particularly limited, but examples thereof include methods using vectors such as viruses, plasmids, and artificial chromosomes, or techniques such as lipofection, liposomes, and microinjection.

[0029] Among the above, it is preferable to use a vector when introducing the polynucleotide of this embodiment into target cells. The vector used in the present invention is not particularly limited as long as it can efficiently deliver genes to target cells (neural stem cells, etc.). Examples of such vectors include viral vectors such as lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, herpesviral vectors (e.g., herpes simplex viral vectors), poxviral vectors, baculoviral vectors, papillomaviral vectors, and papovaviral vectors (e.g., SV40); plasmid vectors; phagemid vectors; cosmid vectors; and bacteriophages such as lambda phage and M13 phage. Of these, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, and plasmid vectors are preferred, with lentiviral vectors and adeno-associated viral vectors being more preferred, and lentiviral vectors being even more preferred.

[0030] Examples of specific expression vectors include pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells; pClneo vector (Promega) for expression in mammalian cells; and the like.

[0031] The vector may contain regulatory sequences such as the promoter and enhancer described above, as well as ribosome binding sequences, terminators, polyadenylation sites, etc., to enable expression of the gene of interest. If necessary, the vector may also contain selectable marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, and diphtheria toxin genes, and reporter gene sequences such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), and FLAG.

[0032] By introducing a vector containing the miR-shRNA of this embodiment and the promoter, etc. into target cells such as neural stem cells, neurons, astrocytes, and oligodendrocytes to transform them, the genes listed in Table 1 above can be knocked down. Target cells such as neural stem cells transformed in this way can be activated and proliferated, and differentiated to fully exert their cellular functions, such as producing a large number of neurons. The vector of this embodiment can be suitably used for neural stem cells, etc., present in adult brains, aging brains, diseased brains, etc.

[0033] (B) siRNA, shRNA, or antisense oligonucleotide against any of the genes listed in Table 1 The inhibitor contained in the preventive and / or therapeutic agent for neurodegenerative disease of the present invention includes siRNA (small interfering RNA), shRNA (small hairpin RNA), antisense oligonucleotide, dsRNA (double-stranded RNA), miRNA (microRNA), etc. against any of the genes listed in Table 1. Of these, siRNA, shRNA, and antisense oligonucleotide are preferred from the viewpoint of the effects of the present invention.

[0034] siRNA is a double-stranded oligo-RNA consisting of an RNA having a sequence complementary to the nucleotide sequence of mRNA of a target gene or a partial sequence thereof (target nucleotide sequence) and its complementary strand. Also included in siRNA are single-stranded RNAs in which a sequence complementary to the target nucleotide sequence (first sequence) is linked to its complementary sequence (second sequence) via a hairpin loop, in which the first sequence forms a double-stranded structure with the second sequence by adopting a hairpin loop structure (shRNA), and dumbbell-shaped nucleic acids in which both ends of the double-stranded structure of the first sequence and the second sequence are closed by loop structures.

[0035] The siRNA may have an overhang at the 5'-end or 3'-end of one or both of the sense strand and the antisense strand. The overhang is formed by adding one to several (e.g., 1, 2, or 3) bases to the end of the sense strand and / or the antisense strand. The base length of the siRNA is not particularly limited as long as it can induce RNA interference, and may be, for example, 10 to 50 bases, 15 to 30 bases, or 21 to 27 bases per strand.

[0036] The siRNA targets the mRNA corresponding to the gene listed in Table 1 above, can be designed based on the base sequence of the mRNA, and the sequence is not particularly limited as long as it can induce RNA interference. The siRNA can be chemically synthesized using known techniques or produced using gene recombination technology.

[0037] shRNA is an RNA that can be degraded by Dicer in vivo to generate siRNA. shRNA has a stem-loop structure containing a double-stranded stem and a hairpin loop. The sequence of this hairpin loop is not particularly limited, but can be 5 to 12 bases (Kawasaki H. et al., Nucleic Acid Res. (2003) 31: 700-707). The shRNA described here refers to shRNA other than the miR-shRNA described in (A) above and is classified as simple-stem-loop-based shRNA. This shRNA is expressed by RNA polymerase III, cleaved by Dicer, and acts. Such shRNAs can be designed and produced based on the gene sequences shown in Table 1 above using methods well known to those skilled in the art.

[0038] The sequence of the antisense oligonucleotide (antisense nucleic acid) is preferably complementary to the target gene or a portion thereof, but does not need to be completely complementary as long as it can effectively suppress gene expression. It is sufficient for the sequence to be preferably 90% or more complementary, more preferably 95% or more complementary, to the transcription product of the target gene. To effectively suppress target gene expression using an antisense nucleic acid, the length of the antisense nucleic acid is preferably 15 bases or more. Furthermore, to avoid nonspecific effects, it is preferable to use multiple antisense nucleic acids complementary to different sequences of the target gene. The antisense nucleic acid of the present invention also includes nucleic acids containing antisense sequences of not only the translated region of the target gene but also the untranslated region.

[0039] The dsRNA has an RNAi effect and is composed of a sense RNA having the same sequence as any contiguous RNA region in the mRNA corresponding to the gene listed in Table 1, and an antisense RNA having a sequence complementary to the sense RNA. The length of the "any contiguous RNA region" is usually 20 to 30 bases, and preferably 21 to 23 bases.

[0040] miRNA refers to a 15-25 base non-coding RNA that is transcribed as a hairpin-like RNA precursor, cleaved by a dsRNA cleaving enzyme with RNase III cleavage activity, incorporated into a protein complex called RISC, and involved in the translational repression of mRNA. In the present invention, miRNA also encompasses precursors of the miRNA (pre-miRNA, pri-miRNA).

[0041] Expression vectors that express these siRNAs (small interfering RNAs), shRNAs (small hairpin RNAs), antisense oligonucleotides, dsRNAs (double-stranded RNAs), miRNAs (microRNAs), and the like can also be used. When used as an expression vector, the vector may contain regulatory sequences such as promoters, enhancers, and terminators to enable expression of the target gene. If necessary, the vector may also contain selectable marker sequences such as drug resistance genes (e.g., kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, and diphtheria toxin genes, and reporter gene sequences such as mCherry (red fluorescent protein), green fluorescent protein (GFP), β-glucuronidase (GUS), and FLAG.

[0042] (C) Antibody Against a Protein Derived from Any of the Genes Listed in Table 1 Above Examples of inhibitors contained in the preventive and / or therapeutic agent for neurodegenerative diseases of the present invention include antibodies against proteins derived from any of the genes listed in Table 1 above. The protein derived from the above genes refers to a protein having an amino acid sequence encoded by the above genes. The inhibitor is preferably an antibody against a protein derived from the above genes or a protein having an amino acid sequence encoded by the above genes, which inhibits, suppresses, or reduces the function of this protein.

[0043] In the present invention, the term "antibody" refers to a protein containing one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and includes a molecule or molecules capable of binding to a specific epitope on an antigen. In the present invention, the "antibody" may be in the form of a full-length immunoglobulin (an antibody having an Fc region and a Fab region, a full-length antibody), F(ab')2, Fab', Fab, Fv antibody (variable fragment of antibody), disulfide-linked Fv (dsFv), single-chain antibody (scFv), or a polymer thereof (e.g., diabody). The antibody may be a monoclonal or polyclonal antibody. The antibody may be, for example, a chimeric antibody, a humanized antibody, or a fully humanized antibody. The antibody may be, for example, a bispecific or oligospecific antibody. These antibodies can be produced by methods conventionally known to those skilled in the art.

[0044] (D) A low-molecular-weight compound having an inhibitory effect on the expression and / or function of any of the genes listed in Table 1 above or proteins derived therefrom.

[0045] Examples of inhibitors contained in the preventive and / or therapeutic agent for neurodegenerative diseases of the present invention include low molecular weight compounds that have an inhibitory effect on the expression and / or function of any of the genes listed in Table 1 above or proteins derived therefrom.

[0046] The above-mentioned low molecular weight compounds can be found from a compound library or the like by, for example, the following screening method.

[0047] A screening method comprising: (1) a step of applying a test substance to neural stem cells and measuring the expression of any of the genes listed in Table 1 above or a protein derived from the gene; and (2) a step of selecting a test substance that has an effect of inhibiting the expression of the gene from the results of step (1).

[0048] The substances selected by the above screening method can efficiently activate and proliferate neural stem cells, producing a large number of neurons. Therefore, it is expected that neural stem cells in the aged brain can be restored to their adolescent or younger state, producing a large number of neurons and improving memory and learning ability. They are also expected to be effective in treating neurodegenerative diseases such as Alzheimer's disease, as they can efficiently activate endogenous neural stem cells, produce a large number of neurons, and improve memory and learning ability.

[0049] [Second Embodiment] A second embodiment of the preventive and / or therapeutic agent for neurodegenerative disease of the present invention is characterized in that it contains, as an active ingredient, a promoter of the expression and / or function of any of the ligand-receptor gene sets listed in Table 2 above, or of the respective proteins derived from any of the gene sets. The ligand-receptor sets listed in Table 2 above are activated downstream of iPaD, as shown in the Examples below. That is, when dormant neural stem cells are activated by iPaD treatment, increased secretion and activation of the corresponding ligands of the ligand-receptor sets in the hippocampus occur, which are expected to act on surrounding cells and suppress amyloid beta (Aβ) deposition. Similar to iPaD treatment, the preventive and / or therapeutic agent for neurodegenerative disease of the present invention contains, as an active ingredient, a substance that promotes (activates) the expression and / or function of the ligand-receptor set, or either of the ligands or receptors.

[0050] The nucleic acid sequences of the genes in Table 2 can be known sequences as the coding region (CDS) of the mRNA sequence of each of the above genes, and for example, the transcript sequence of each gene registered in Ensembl, a nucleotide sequence database, can be used. The nucleic acid sequences of the genes in Table 2 in the present invention are identical to, or have 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more homology to, the transcript sequences of the genes registered in Ensembl, and the peptides encoded by these sequences have the activity corresponding to each gene registered in the Ensembl nucleotide sequence database.

[0051] The genes listed in Table 2 above are derived from mammals such as humans, monkeys, pigs, horses, cows, rabbits, sheep, goats, cats, dogs, guinea pigs, and mice, and are preferably derived from humans, monkeys, or mice, with humans being more preferred.

[0052] The promoter is not particularly limited as long as it promotes the expression and / or function of any of the genes listed in Table 2 above or a protein derived from any of the genes.

[0053] [Usage, dosage, specific target diseases, etc. of the preventive and / or therapeutic agent for neurodegenerative disease] The preventive and / or therapeutic agent for neurodegenerative disease of the present invention is administered to a subject by lumbar puncture, intraventricular injection, intrathecal bolus injection or infusion, intraganglionic injection, intraneuronal injection, or subcutaneous injection.

[0054] The dosage of the agent for preventing and / or treating neurodegenerative diseases of the present invention is not particularly limited, and an appropriate dosage can be selected depending on various conditions such as the type of disease, the age and symptoms of the patient, the route of administration, the purpose of treatment, and the presence or absence of concomitant medications.

[0055] Specific diseases for which the agent for preventing and / or treating neurodegenerative diseases of the present invention is effective include Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia caused by frontotemporal lobar degeneration, etc. Among these, the agent for preventing and / or treating neurodegenerative diseases of the present invention is preferably used for Alzheimer's disease and Parkinson's disease, from the viewpoint of obtaining a greater effect.

[0056] <Method of Treatment> The present invention also includes a method of preventing and / or treating a neurodegenerative disease, characterized by using an inhibitor of any of the genes listed in Table 1 above or a protein derived from any of the genes, for the prevention and / or treatment of the neurodegenerative disease. The present invention also includes a method of preventing and / or treating a neurodegenerative disease, characterized by using a promoter of the expression and / or function of any of the ligand-receptor gene sets listed in Table 2 above or each protein derived from any of the gene sets, for the prevention and / or treatment of the neurodegenerative disease.

[0057] The methods for preventing and / or treating neurodegenerative diseases of the present invention are as follows: [1] A method for preventing and / or treating neurodegenerative diseases, comprising administering an inhibitor of any of the genes listed in Table 1 above or a protein derived from any of the genes. [2] The method for preventing and / or treating neurodegenerative diseases according to [1], wherein the inhibitor is: (A) a vector having a polynucleotide comprising a nucleic acid sequence of a microRNA adapted short hairpin RNA (miR-shRNA) for any of the genes listed in Table 1 above and a promoter sequence operably linked to the nucleic acid sequence; (B) an siRNA, shRNA, or antisense oligonucleotide for any of the genes listed in Table 1 above; (C) an antibody against a protein derived from any of the genes listed in Table 1 above; or (D) a small molecule compound having an inhibitory effect on the expression and / or function of any of the genes listed in Table 1 above or a protein derived therefrom. [3] The method for preventing and / or treating neurodegenerative diseases according to [1] or [2], wherein the gene is a mammalian gene. [4] The method for preventing and / or treating a neurodegenerative disease according to [2] or [3], wherein the vector is a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a plasmid vector. [5] The method for preventing and / or treating a neurodegenerative disease according to any of [1] to [4], wherein the vector is administered by lumbar puncture. [6] A method for preventing and / or treating a neurodegenerative disease, comprising administering a promoter for the expression and / or function of any of the ligand-receptor gene sets listed in Table 2 above, or a protein derived from any of the gene sets. [7] The method for preventing and / or treating a neurodegenerative disease according to any of [1] to [6], wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, and dementia caused by frontotemporal lobar degeneration. [8] The method for preventing and / or treating a neurodegenerative disease according to any of [1] to [6], wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease and Parkinson's disease.[9] The method for preventing and / or treating a neurodegenerative disease according to any one of [1] to [6], wherein the neurodegenerative disease is Alzheimer's disease.

[0058] For a specific description of the agent for preventing and / or treating neurodegenerative diseases of the present invention, the description in the section on agent for preventing and / or treating neurodegenerative diseases can be applied as is.

[0059] <Method for screening for substances for preventing and / or treating neurodegenerative diseases> A first embodiment of the present invention is a method for screening for substances for preventing and / or treating neurodegenerative diseases, comprising: (1) a step of allowing a test substance to act on neural stem cells, and measuring the expression of any of the genes listed in Table 1 above, or the expression and / or activity of a protein derived from the gene; and (2) a step of selecting, from the results of step (1), a test substance that has an effect of suppressing the expression of the gene, or an effect of suppressing the expression or activity of a protein derived from the gene.

[0060] As the neural stem cells, in addition to primary cultured cells of neural stem cells derived from various animals, neural stem cells differentiated from stem cells such as ES cells or iPS cells using well-known methods, and cell lines such as mouse neural stem cell lines such as MEB5 may be used.

[0061] The test substance in the present invention is not particularly limited, and examples thereof include single substances such as natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins (including antibodies), and peptides; expression products of compound libraries, nucleic acid libraries, peptide libraries, and gene libraries; cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single-cell extracts, and animal cell extracts. The test substance in the present invention may also be a mixture of these.

[0062] The method for measuring the expression of any of the genes listed in Table 1 above or the expression and / or activity of a protein derived from the gene may be any of the conventionally known methods for measuring gene expression, protein expression, or protein activity in various cells. Examples include a method in which the expression and / or activity of each gene or protein is measured in the presence and absence of a test substance, and the result is determined based on the difference between the presence and absence of the test substance or the ratio between the two. Using these methods, a test substance that exhibits an effect of suppressing the expression of the gene or the expression or activity of a protein derived from the gene is selected.

[0063] A second embodiment of the present invention is a method for screening for a substance for preventing and / or treating a neurodegenerative disease, comprising: (i) a step of allowing a test substance to act on neural stem cells, and measuring the expression of any of the genes listed in Table 2 above, or the expression and / or activity of a protein derived from the gene; and (ii) a step of selecting, from the results of step (i), a test substance that has an effect of promoting the expression of the gene, or an effect of promoting or activating the expression of a protein derived from the gene.

[0064] The explanation in the first embodiment is applicable to the neural stem cells and the test substance.

[0065] The method for measuring the expression of any of the genes listed in Table 2 above, or the expression and / or activity of a protein derived from the gene, may be any of the conventionally known methods for measuring gene expression, protein expression, or protein activity in various cells. Examples include a method in which the expression and / or activity of each gene or protein is measured in the presence and absence of a test substance, and the result is determined based on the difference between the presence and absence of the test substance, or the ratio between the two. Using these methods, a test substance that has the effect of promoting the expression of the gene or the effect of promoting the expression or enhancing the activity of a protein derived from the gene is selected.

[0066] The substances selected by the above screening method can efficiently activate and proliferate neural stem cells, producing a large number of neurons. Therefore, it is expected that neural stem cells in the aged brain can be restored to their adolescent or younger state, producing a large number of neurons and improving memory and learning ability. They are also expected to be effective in treating neurodegenerative diseases such as Alzheimer's disease, as they can efficiently activate endogenous neural stem cells, produce a large number of neurons, and improve memory and learning ability.

[0067] The present disclosure will be specifically described in the following examples, but the present disclosure should not be construed as being limited by these examples.

[0068] The materials and methods used in this example are as follows. Animals: 5XFAD mice, a mouse model of Alzheimer's disease (Oakley et al., 2006), were obtained from the Jackson Laboratory. Briefly, 5XFAD mice harbor a double transgenic construct of mutant human amyloid precursor protein (APP) (Swedish mutation: K670N, M671L; Florida mutation: I716V; London mutation: V717I) and presenilin 1 (PS1) (M146L; L286V) under the control of the mouse Thy1 promoter (Tg6799 strain). The 5xFAD strain (B6 / SJL genetic background) was obtained from the Jackson Laboratory and backcrossed with C57BL / 6J wild-type (WT) mice for more than two generations. WT littermate mice served as controls. All mice were handled in accordance with the Kyoto University Guidelines for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Experimental Animal Committee of the Institute for Virus Research and Frontier Medical Sciences, Kyoto University.

[0069] Lentivirus Construction: Lentiviruses were produced by subcloning each gene sequence into the CSII-1.6-kb-pmHes5-mCherry-3NLS-MCS or CSII-pEFS-mCherry-3NLS-MCS plasmid. For lentivirus production, 84 μg of each CSII plasmid, 36.5 μg of the gag-pol plasmid (psPAX2), and 11.7 μg of the VSV-G plasmid (pMD2.G) were co-transfected into five 15 cm dishes of HEK293 cells using 860 μL of 1 mg / ml polyethyleneimine-MAX (Polysciences). Cells were cultured in OptiPRO serum-free medium (Gibco) containing 10 μM forskolin, 1 mM sodium pyruvate, and GlutaMax (Thermo) for 3 days, after which the medium was harvested. A total of 100 mL of medium was concentrated in 100 μL of PBS by centrifugation at 8,000 × g for 12 hours and 13,000 × g for 4 hours. Lentiviral titers were measured by infecting cultured NSCs, typically reaching 2 × 10 7 Titers of units / ml were obtained. Lentiviral vectors with miR-E backbones were used for gene expression knockdown. The sequences shown in the table below were used for knockdown. A scrambled sequence was used as a negative control.

[0070]

[0071] Lentiviral infection in mice: For in vivo screening and neurogenesis induction, 1 μL (3.75 × 10 5 The dentate gyrus was stereotactically delivered at a flow rate of 0.125 μL / min to the following coordinates: anteroposterior = -2.0 mm, lateral = ±1.4 mm, ventral = 2.0 mm from bregma. Brain sections were subsequently examined immunohistochemically.

[0072] Immunohistochemical Analysis: For immunohistochemical analysis of brain tissue, mice were perfused transcardially with 4% paraformaldehyde in PBS. Brains were then postfixed in 4% paraformaldehyde in PBS, cryoprotected by sequential overnight incubations in 30% sucrose in PBS, embedded, and frozen in OCT (Tissue TEK). Serial 50-μm-thick coronal cryosections were obtained using a cryostat. Primary antibodies used were rabbit anti-doublecortin (1:500; Cell Signaling Technology 14802), rabbit anti-MCM2 (1:500; Abcam ab4461), rat anti-mCherry (1:500; Invitrogen M11217), and mouse anti-β-amyloid (clone 6E10; 1:500; Biolegend 9320). Primary antibodies were incubated overnight, followed by secondary antibodies overnight.

[0073] Quantification of Labeled Cells. The DG-SGZ was analyzed in at least three mice per experiment. The DG region of each mouse was evaluated every seventh section. Immunostaining was performed on all sections except those damaged for labeling. Z-stack images were then captured using a confocal microscope (LSM780 or LSM980) with a 10x or 20x objective. Stained cells were quantified using ImageJ and expressed as the number of cells per mm3 of the DG-SGZ in each image.

[0074] Quantification of Amyloid-β (Aβ) Deposition. Every seventh brain section from each mouse was labeled with a mouse anti-Aβ primary antibody (clone 6E10; 1:500; BioLegend 9320) and detected with a fluorescently labeled secondary antibody. After immunostaining, z-stack images were acquired using a confocal microscope (LSM780 or LSM980) at a 10x or 20x objective. The number of Aβ plaques was quantified using ImageJ after appropriate thresholding and despeckling. Results were expressed as plaque coverage per mm3 of DG-SGZ in each image.

[0075] Preparation of Dentate Gyrus Tissue from Lentivirus-Injected 5XFAD Mice and Transcriptome Analysis: Four weeks after lentivirus infection of 5-month-old 5XFAD mice, total RNA was isolated from DG tissue using the RNeasy Mini Kit (Qiagen). RNA quality was assessed using an RNA Bioanalyzer, and all samples were confirmed to have an RNA Integrity Number (RIN) of 9 or higher. Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Paired-end sequencing was performed using a HiSeq X (Illumina). cDNA sequences were aligned to the mouse reference genome mm10, counted using STAR, and normalized using edgeR with the TPM normalization method. Outlier samples were excluded from the analysis. Gene expression and GO enrichment analyses were performed using edgeR and Metascape. The ligand-receptor pair list was obtained from CellTalkDB (Shao et al. 2021).

[0076] Statistical analysis: Data are presented as mean ± SEM. Statistical analysis was performed using R software. Statistical differences were determined by one-way and two-way analysis of variance, followed by post-hoc comparison tests using the Tukey-Kramer and Bonferroni methods. A P value of <0.05 was considered statistically significant.

[0077] 1. iPaD Enhances Neurogenesis and Suppresses Aβ Deposition. To clarify the impact of activated neurogenesis on Alzheimer's disease, we injected control lentivirus or iPaD lentivirus (combined Plagl2 overexpression and Dyrk1a knockdown; see Non-Patent Document 6 for details) into the hippocampal dentate gyrus of 5-month-old 5XFAD mice under the control of the Hes5 promoter, which is activated in neural stem cells (NSCs). The hippocampal regions were examined at set times (4, 8, or 12 weeks post-infection (wpi)). Lentivirus-infected NSCs and their progeny became mCherry+ (Figure 1). Control lentivirus-injected 5XFAD mice showed significantly reduced neurogenesis, with very few activated NSCs (MCM2+, Figure 1A, control) or DCX+ neuroblasts (Figure 1B, control) at all stages of the study. In contrast, iPaD lentivirus-injected 5XFAD mice exhibited abundant activated NSCs (MCM2+, Fig. 1A, iPaD) and DCX+ neuroblasts (Fig. 1B, iPaD) at all stages examined, indicating significant NSC activation and sustained enhancement of neurogenesis.

[0078] We also investigated the effect of iPaD treatment on Aβ pathology. In control lentivirus-injected 5XFAD mice, Aβ deposition increased with age (Figure 2, control). In contrast, in iPaD lentivirus-injected 5XFAD mice, Aβ deposition did not increase with age (Figure 2, iPaD). These results suggest that iPaD treatment efficiently activates NSCs, promotes neurogenesis, and reduces Aβ deposition. The combined effect of Plagl2 overexpression and Dyrk1a knockdown has been termed "iPaD (inducing Plagl2 and anti-Dyrk1a)."

[0079] 2. Downstream events of iPaD To understand the mechanism by which iPaD treatment improves the AD phenotype in NSCs, we injected iPaD lentivirus into the hippocampal dentate gyrus of 5-month-old 5XFAD mice. Four weeks later, we isolated the hippocampus and performed RNA-seq analysis. The results showed that iPaD-treated NSCs significantly upregulated 592 genes and downregulated 462 genes throughout the hippocampus of 5XFAD mice (Fig. 3, p-value <0.05).

[0080] Gene ontology (GO) analysis of the up-regulated genes revealed that they included genes involved in myelination, immunity, cytokines, and microglia (Fig. 4A). Ligand-receptor pairs listed in CellTalkDB (http: / / tcm.zju.edu.cn / celltalkdb / ) were extracted from these genes and identified as up-regulated downstream of iPaD treatment, including Apoe-Abca1 / Trem2, Il33-Il1rl1, Ccl3-Ccr5, C3 / Icam1-Itgb2, and Lgals3-Mertk, as shown in Table 4. These ligand-receptor pairs are known to ameliorate the pathology of Alzheimer's disease (Salta E.et al., 2023. Adult hippocampal neurogenesis in Alzheimer's disease: a road map to clinical relevance. Cell Stem Cell 30: 120-136), so these results suggest that iPaD treatment ameliorates the pathology of Alzheimer's disease by activating these ligand-receptor pairs.

[0081]

[0082] GO term analysis of the downregulated genes revealed that they included genes involved in cell morphogenesis, extracellular matrix (ECM), and development (Figure 4B). While several ECM proteins are known to be involved in Alzheimer's disease pathology (Sun et al., 2021, Role of the extracellular matrix in Alzheimer's disease. Front Aging Neurosci 13: 707466), the functional significance of most other genes has not been characterized. Therefore, we decided to examine whether knockdown of these genes would ameliorate Alzheimer's disease pathology using the miR-E backbone shRNA system (Fellmann et al., 2013, An optimized microRNA backbone for effective single-copy RNAi. Cell Rep 5: 1704-1713). The knockdown strategy is shown in Figure 5.

[0083] 3. Increased neurogenesis and reduced Aβ deposition by knockdown of Prkag2, Gfra2, Rpl29, and Maml2 The table below shows the list of genes whose expression was most significantly decreased by iPaD treatment in the hippocampus of 5XFAD mice.

[0084]

[0085] Among the genes listed above, Maml2, Rpl29, Ptgds, Ell3, Prkag2, Cspp1, Gfra2, Itpk1, and Mical3 were selected as the most significantly downregulated genes by iPaD treatment, and the overall knockdown effect on Aβ pathology was examined. Using lentivirus, expression of the miR-E backbone shRNA (SEQ ID NOs: 1 to 8 in Table 3) for each gene was induced together with mCherry under the control of the ubiquitous elongation factor promoter in the hippocampus of 5XFAD mice, and the effect was examined 8 weeks later (Figure 5).

[0086] Among the genes examined, knockdown of Prkag2, encoding the protein kinase AMP-activated non-catalytic subunit γ2; Gfra2, encoding the glial cell line-derived neurotrophic factor family receptor α2; and Rpl29, encoding a ribosomal protein, more effectively increased the number of DCX+ neuroblasts (Fig. 6A, B) and suppressed Aβ deposition (Fig. 7A, B). Knockdown of Maml2, encoding a coactivator of Notch signaling, also increased the number of DCX+ neuroblasts (Fig. 6A, B) and suppressed Aβ deposition (Fig. 7A, B), but was less effective than Prkag2. These results suggest that knockdown of Prkag2, Gfra2, Rpl29, and Maml2 effectively stimulates neurogenesis and ameliorates Aβ pathology, suggesting that these genes may be novel targets for the treatment of Alzheimer's disease.

[0087] As described above, we found that many genes involved in cell morphogenesis, extracellular matrix (ECM), and developmental growth were downregulated by iPaD treatment, and among them, downregulation of Prkag2, Gfra2, Rpl29, and Maml2 was important for activating neurogenesis and suppressing Aβ accumulation.

[0088] 4. Elucidation of the mechanism by which Prkag2 knockdown increases neurogenesis and reduces Aβ deposition. We focused on Prkag2, which showed the most significant increase in neurogenesis and reduction in Aβ deposition upon knockdown, and explored its mechanism of action. Prkag2 is one of the subunits (γ subunit 2) of AMP-activated protein kinase (AMPK). AMPK signaling is known to activate autophagy to degrade amyloid-β deposits, reduce lipid droplets, and activate microglial phagocytosis. Through these actions, AMPK signaling is thought to ameliorate the pathology of Alzheimer's disease. Measurement of the amount of activated AMPK (phosphorylated Thr172 of the AMPK α subunit = pAMPK) revealed that 4-month-old 5xFAD mice had similar levels of pAMPK-positive cells and lipid droplets (BODIPY+) compared to wild-type mice (Figure 8, 4M WT and 4M 5xFAD). However, at 8 months of age, 5xFAD mice showed a significant decrease in pAMPK-positive cells and an increase in lipid droplets (Fig. 8, 8M 5xFAD). Furthermore, amyloid-β deposition was also significantly increased in 5xFAD mice at this age. These findings suggest that a decrease in AMPK signaling contributes to the worsening of Alzheimer's disease pathology, such as amyloid-β deposition and an increase in lipid droplets.

[0089] Next, we analyzed the effects of Prkag2 knockdown in neural stem cell cultures and found an increase in AMPK activity (Figure 9). To clarify why Prkag2 knockdown increases AMPK activity, we examined the expression of other AMPK subunits using qPCR. We found that Prkag2 knockdown significantly increased the expression of many subunits (catalytic subunits Prkaa1 and Prkaa2, structural subunit Prkab1, and regulatory subunit Prkkag1) (Figure 10), which may contribute to increased AMPK activity. Therefore, Prkag2 knockdown may ameliorate the pathology of Alzheimer's disease by activating AMPK signaling.

[0090] Furthermore, we investigated the effect of Prkag2 knockdown on the proliferation of neural stem cells in culture. Although there was no significant difference, Prkag2 knockdown tended to increase EdU incorporation (Fig. 11). Therefore, it is suggested that Prkag2 knockdown directly activates neural stem cells and activates their proliferation. In contrast, Maml2 knockdown did not show such an effect (Fig. 11).

[0091] Finally, we explored the molecular mechanism by which iPaD-activated neural stem cells activate AMPK signaling in the peripheral region. Based on the findings that iPaD-activated neural stem cells and the newborn neurons they differentiate from secrete AMPK activators, we searched for secreted factors and receptors involved in AMPK signaling among the genes activated by iPaD. As a result, we identified Adipoq-Adipor2, Il33-Il1rl1, and Il17a-Il17rc (Figure 12). Furthermore, when iPaD was applied to neural stem cell cultures, the expression of Adipoq, Il33, and Il17a increased (Figure 13). Therefore, it is possible that iPaD increases the production of Adipoq, Il33, and Il17a in neural stem cells, activating AMPK signaling in the peripheral region and ameliorating amyloid pathology.

[0092] Prkag2 is a regulatory subunit of AMP-activated kinase (AMPK), which regulates cellular energy metabolism. It has been reported that Prkag2 expression is upregulated in postmortem brains of Alzheimer's disease patients (Bharadwaj and Martins 2020, PRKAG2 gene expression is elevated and its protein levels are associated with increased Amyloid-β accumulation in the Alzheimer's disease brain. J Alzheimers Dis 74: 441-448). However, it was unclear whether this upregulation contributes to Aβ pathology or simply serves to alleviate the pathology. We found that Prkag2 knockdown in 5XFAD mice most effectively suppressed Aβ deposition, suggesting that downregulation of Prkag2 expression may ameliorate Aβ pathology not only in mice but also in humans. We also found that Prkag2 knockdown effectively stimulates neurogenesis in 5XFAD mice, although the underlying mechanism remains unclear. It has been reported that suppression of AMPKα1, the catalytic subunit of AMPK, alleviates cognitive impairment in Alzheimer's disease model mice (Zimmermann et al., 2020, Brain-specific repression of AMPKα1 alleviates pathophysiology in Alzheimer's model mice. J Clin Invest 130: 3511-3527). Furthermore, knockdown of AMPK has been shown to protect against amyotrophic lateral sclerosis model animals (Lim et al., 2012, Reduced activity of AMP-activated protein kinase protects against genetic models of motor neuron disease. J Neurosci 32: 1123-1141).These data suggest that modulation of AMPK signaling may be important for the treatment of various neurodegenerative diseases, including Alzheimer's disease.

[0093] Gfra2 is upregulated in postmortem brains of patients with Alzheimer's disease (Haytural et al., 2021, Insights into the changes in the proteome of Alzheimer's disease elucidated by a meta-analysis. Sci Data 8: 312), but its role in Alzheimer's disease is unclear. Its ligand, glial cell line-derived neurotrophic factor (GDNF), has been shown to regulate axonal growth and neuronal survival, and neurons from patients with Alzheimer's disease have been shown to lack a response to GDNF (Konishi et al., 2014, Deficiency of GDNF receptor GFRα1 in Alzheimer's neurons results in neuronal death. J Neurosci 34: 13127-13138). GDNF concentrations in serum were significantly lower and those in CSF were significantly higher in patients with Alzheimer's disease compared with age-matched controls (Straten et al., 2009, Glial cell-line derived neurotrophic factor (GDNF) concentrations in cerebrospinal fluid and serum of patients with early Alzheimer's disease and normal controls. J Alzheimers Dis 18: 331-337.). These results suggest that upregulation of GDNF in CSF may be an adaptive response to enhance neurotrophic support. Because GDNF selectively binds to a different receptor subtype, Gfra1, knockdown of Gfra2 may facilitate the interaction between GDNF and Gfra1.However, Gfra2-deficient mice exhibit behavioral and memory impairments (Voikar et al., 2004, Impaired behavioral flexibility and memory in mice lacking GDNF family receptor α2. Eur J Neurosci 20: 308-312), suggesting that a certain level of Gfra2 expression is required for normal brain function. Further analysis is needed to determine how Gfra2 knockdown ameliorates Aβ pathology. Because GDNF is upregulated by AMPK signaling (Katila et al., 2020), knockdown of Prkag2 and Gfra2 may ameliorate Aβ pathology by modulating AMPK-GDNF pathway activity.

[0094] Maml2 proteins are a family of three coactivators for various transcription factors, including the Notch signaling factor RBPj (Lin et al., 2002, Identification of new human mastermind proteins defines a family that consists of positive regulators for notch signaling. J Biol Chem 277: 50612-50620; Wu et al., 2002, Identification of a family of mastermind-like transcriptional coactivators for mammalian notch receptors. Mol Cell Biol 22: 7688-7700) and myocyte enhancer factor 2C (Shen et al., 2006, The Notch coactivator, MAML1, functions as a novel coactivator for MEF2C-mediated transcription and is required for normal myogenesis. Genes Dev 20: 675-688). Therefore, knockdown of Maml2 is expected to downregulate the expression of many genes, including those involved in Notch signaling. Here, we found that knockdown of Maml2 effectively stimulates neurogenesis and reduces Aβ deposition. In the adult brain, NSC quiescence is controlled by high expression of the Notch effector Hes1 (Sueda et al., 2019, High Hes1 expression and resultant Ascl1 suppression regulate quiescent vs. active neural stem cells in the adult mouse brain. Genes Dev 33: 511-523), and knockdown of Maml2 may downregulate Hes1 expression, leading to NSC activation.However, the mechanism by which Maml2 knockdown reduces Aβ deposition has not yet been elucidated. Recently, it has been shown that Maml2 is upregulated in human Alzheimer's disease patients (Xiong et al., 2023, Epigenomic dissection of Alzheimer's disease pinpoints causal variants and reveals epigenome erosion. Cell 186: 4422-4437), suggesting that Maml2 knockdown may be effective in reducing Aβ pathology not only in mice but also in humans.

[0095] Knockdown of Prkag2, Gfra2, Maml2, and / or Rpl29 activated adult neurogenesis and reduced Aβ deposition, and we noted that the degree of activated neurogenesis correlated well with the degree of reduced Aβ deposition. The activation of neurogenesis may reduce Aβ deposition, and the reduction of Aβ deposition may activate neurogenesis. Activation of adult neurogenesis has been shown to improve age-related cognitive impairment and neurodegenerative diseases (Benraiss et al., 2013, Mobilization of endogenous progenitor cells regenerates functionally integrated medium spiny striopallidal projection neurons and delays disease progression in a transgenic model of Huntington's disease. Cell Stem Cell 12: 787-799; Choi et al., 2018, Combined adult neurogenesis and BDNF mimic exercise effects on cognition in an Alzheimer's mouse model. Science 361: eaan8821; Diaz-Moreno et al., 2018, Noggin rescues age-related stem cell loss in the brain of senescent mice with neurodegenerative pathology. Proc Natl Acad Sci USA 115: Knockdown of Prkag2, Gfra2, Maml2, and / or Rpl29 has shown promise as a therapeutic strategy for various brain disorders, including Alzheimer's disease, as it can improve the neuroprotective effects of cerebrospinal fluid (e.g., cerebrospinal fluid, cerebrospinal fluid, and cerebrospinal fluid).

[0096] According to the present invention, suppressing or inhibiting the expression of the specific genes or proteins derived from any of these genes can efficiently activate neurogenesis in the adult brain and reduce amyloid-β deposition. Such activation of neurogenesis and reduction of amyloid-β deposition in the adult brain are thought to lead to improvements in age-related cognitive dysfunction and neurodegenerative diseases. Therefore, knockdown of the specific genes, such as Prkag2, Gfra2, Maml2, and Rpl29, or proteins derived from any of these genes, is strongly suggested to be a promising therapeutic strategy for neurodegenerative diseases, including Alzheimer's disease.

Claims

1. A prophylactic and / or therapeutic agent for neurodegenerative diseases, comprising, as an active ingredient, an inhibitor of any of the genes described in Table 1 below, or a protein derived from any of the genes.

2. The preventive and / or therapeutic agent for neurodegenerative diseases according to claim 1, wherein the inhibitor is (A) a vector having a polynucleotide containing a nucleic acid sequence of miR-shRNA (microRNA adapted short hairpin RNA) for any one of the genes described in Table 1 above and a promoter sequence operably linked to the nucleic acid sequence, (B) siRNA, shRNA, or an antisense oligonucleotide for any one of the genes described in Table 1 above, (C) an antibody against a protein derived from any one of the genes described in Table 1 above, or (D) a low molecular weight compound having an inhibitory effect on the expression and / or function of any one of the genes described in Table 1 above or a protein derived therefrom.

3. The preventive and / or therapeutic agent for neurodegenerative diseases according to claim 1 or 2, wherein the gene is a mammalian gene.

4. The preventive and / or therapeutic agent for neurodegenerative diseases according to claim 2 or 3, wherein the vector is a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a plasmid vector.

5. The preventive and / or therapeutic agent for neurodegenerative diseases according to any one of claims 1 to 4, which is administered by lumbar puncture.

6. A prophylactic and / or therapeutic agent for neurodegenerative diseases, comprising, as an active ingredient, an agent that promotes the expression and / or function of any gene set of ligand-receptor described in Table 2 below, or a protein derived from any gene set.

7. The preventive and / or therapeutic agent for neurodegenerative diseases according to any one of claims 1 to 6, wherein the neurodegenerative disease is at least one selected from the group consisting of dementia caused by Alzheimer's disease, Parkinson's disease, Huntington's disease, and frontotemporal lobar degeneration.

8. The preventive and / or therapeutic agent for neurodegenerative diseases according to any one of claims 1 to 6, wherein the neurodegenerative disease is at least one selected from the group consisting of Alzheimer's disease and Parkinson's disease.

9. The preventive and / or therapeutic agent for neurodegenerative diseases according to any one of claims 1 to 6, wherein the neurodegenerative disease is Alzheimer's disease.

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