Agent for preventing or treating dementia

Abstract

The present invention provides a preventive or therapeutic agent for dementia, an enhancer of memory and / or cognitive function, an agent for inhibiting the synapse-eliminating action of amyloid-beta protein, an amyloid-beta protein production inhibitor, and an amyloid-beta protein clearance promoter, which contain as an active ingredient the Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof.

Description

Technical Field

[0001] The present invention relates to a preventive or therapeutic agent for dementia and an enhancer for memory and / or cognitive function using the Crtac1B protein or a C-terminal region fragment thereof.

Background Art

[0002] Even in healthy individuals, it is known that with aging, slow inflammatory effects and brain atrophy occur, gradually weakening the neuroplasticity responsible for memory and learning, and as a result, symptoms such as senile amnesia may appear. On the other hand, in dementia typified by Alzheimer's disease, it is known that various higher brain functions including memory and learning are impaired by nerve cell death induced by the accumulation of amyloid-β (Aβ) protein and phosphorylated tau.

[0003] Since the former is not a pathological condition, there is no therapeutic agent for it, and various health methods and functional foods have been devised, but their effects have not been academically recognized. In the latter case, drugs that increase the amount of neurotransmitter (acetylcholine) in synapses, typified by Aricept, by inhibiting its degradation, and drugs that protect against cell death caused by excessive excitation of nerve cells by NMDA receptor antagonists such as memantine have been applied as limited symptomatic therapies, but their usefulness has not been widely recognized. Furthermore, many inhibitors against two enzymes (BACE1, γ-secretase; which cleave amyloid precursor protein (APP) to produce Aβ) responsible for Aβ production have also been developed, but they have been unable to inhibit the accumulation of Aβ, and research targeting these enzymes has declined.

[0004] Recently, lecanemab and donanemab have been developed as antibody pharmaceuticals aiming at fundamental improvement of Aβ pathology and have obtained regulatory approval. Although the usefulness of antibodies against Aβ was proposed nearly half a century ago (Non-Patent Document 1), the development was aborted due to deaths (deaths due to severe encephalitis) in Aβ vaccine therapy. As a result of long years of research and development with the progress of anti-Aβ antibody drug development, lecanemab has emerged. However, although these antibody pharmaceuticals have been confirmed to suppress the decline of cognitive function in dementia patients, their effects are not sufficient, and it is difficult to improve the already declined cognitive function if the treatment intervention time is late, so the effects are limited. The efficacy and effect of lecanemab approved in Japan is limited to mild cases of "suppression of progression of mild cognitive impairment and mild dementia due to Alzheimer's disease" (Non-Patent Document 2). Also in the United States, the clinical trial of lecanemab was conducted targeting patients with early Alzheimer's disease (Non-Patent Documents 3, 4), and administration is to be started from mild patients. In addition, antibody pharmaceuticals such as lecanemab require continuous administration over a long period, and there are concerns about serious side effects such as cerebral edema at present, and there are numerous unsolved problems.

[0005] In addition, as a countermeasure against tau pathology, intrathecal administration of antisense oligos as nucleic acid pharmaceuticals that reduce tau has been attempted (Non-Patent Document 5), and antibody pharmaceuticals that prevent tau existing intracellularly from being secreted extracellularly and spreading in the brain are also being studied. These pharmaceuticals are the world's most advanced technologies for dementia, but many years are still required for clinical application.

[0006] Thus, although pharmaceutical development targeting Aβ and tau is progressing, as described above, the mainstream of development is the approach using antibodies against those target toxic substances and the approach aiming at reduced expression by nucleic acid pharmaceuticals. The control method by endogenous substances is undeveloped.

[0007] The group of the inventors of the present application has previously discovered an endogenous protein that forms a neural circuit responsible for olfactory information and named it LOTUS (Lateral Olfactory Tract Usher Substance) (Non-Patent Document 3). LOTUS is Crtac1B, one of the splicing variants of Crtac1, which is known as a marker for chondrocyte progenitor cells, and is mainly expressed in the nervous system. LOTUS / Crtac1B binds to Nogo receptor type 1 (NgR1) and PirB, among the receptors that function in the nerve regeneration inhibitory pathway, blocks ligand binding, and promotes nerve regeneration by inhibiting signal transduction by these receptors (Non-Patent Documents 6 and 7).

[0008] Regarding the association between LOTUS / Crtac1B and neurodegenerative diseases of the brain, it has been reported that synaptic density decreases in cultured hippocampal neurons lacking LOTUS / Crtac1B, and that social cognitive memory and spatial-dependent memory in mice are impaired by the deficiency of LOTUS / Crtac1B (Non-Patent Document 8). However, the effects of replenishing LOTUS / Crtac1B on the cognitive and memory functions of the living body have not been reported. The phagocytosis of accumulated Aβ and the action of LOTUS / Crtac1B on Aβ production are also completely unknown.

Prior Art Documents

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0010] An object of the present invention is to provide a novel means effective for preventing dementia such as Alzheimer's disease and memory and cognitive function decline due to aging, and improving memory and cognitive function. [Means for Solving the Problems]

[0011] As a result of intensive research, the inventors of the present application have found that LOTUS / Crtac1B binds to the enzyme BACE1 responsible for Aβ production, suppresses the activity of BACE1, inhibits APP cleavage by BACE1 (inhibition of Aβ production), improves the survival rate of microglia, and promotes the phagocytosis of Aβ by microglia (promotion of Aβ clearance). They have also found that it inhibits the binding of Aβ and PirB and suppresses the decrease in spine density caused by Aβ (suppression of synapse loss caused by Aβ). Furthermore, LOTUS supplementation by overexpressing LOTUS increases the spine density and synapse density of mouse hippocampal neurons, enhances social cognitive memory and spatial learning memory in young and old brains (enhancement of cognitive and memory functions in non-Alzheimer's brains), and improves memory impairment in Alzheimer's disease model mice (enhancement of cognitive and memory functions in Alzheimer's disease). Based on these findings, the present invention has been completed.

[0012] That is, the present invention is an invention for treating dementia, preventing or improving the decline in cognitive and memory functions due to aging, etc., by supplementing a patient with the LOTUS / Crtac1B protein or a C-terminal region fragment of LOTUS / Crtac1B, which is a functional domain that exhibits an antagonistic effect against NgR1, and includes the following aspects.

[0013] [1] A prophylactic or therapeutic agent for dementia, containing as an active ingredient the Crtac1B protein or its C-terminal region fragment, a vector capable of expressing the Crtac1B protein or its C-terminal region fragment, or a cell expressing the Crtac1B protein or its C-terminal region fragment. [2] The prophylactic or therapeutic agent according to [1], wherein the dementia is Alzheimer's type dementia. [3] An enhancer of memory and / or cognitive function, containing as an active ingredient the Crtac1B protein or its C-terminal region fragment, a vector capable of expressing the Crtac1B protein or its C-terminal region fragment, or a cell expressing the Crtac1B protein or its C-terminal region fragment. [4] An agent that inhibits the synapse-loss effect of amyloid-beta protein, containing as an active ingredient Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing Crtac1B protein or a C-terminal region fragment thereof. [5] An amyloid-beta protein production inhibitor, containing as an active ingredient Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing Crtac1B protein or a C-terminal region fragment thereof. [6] An amyloid-beta protein clearance promoter, containing as an active ingredient Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing Crtac1B protein or a C-terminal region fragment thereof. [7] The Crtac1B protein is a polypeptide having an amino acid sequence with 95% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and the C-terminal region fragment is (i) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 7, (ii) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 9, (iii) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 11, or (iv) A polypeptide having 90% or more sequence identity to the protein fragments of (i) to (iii) The agent according to any one of [1] to [6]. [8] The agent according to any one of [1] to [7], wherein the cell expressing Crtac1B protein or a C-terminal region fragment thereof is a neural stem / progenitor cell, an excitatory neuron or an oligodendrocyte progenitor cell. [9] A method for preventing or treating dementia, comprising administering to a dementia patient a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof.

[10] The method according to [9], wherein the dementia is Alzheimer's disease.

[11] A method for enhancing memory and / or cognitive function, comprising administering to a patient in need of enhancing memory and / or cognitive function a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof.

[12] The method according to

[11] , wherein the patient is an elderly person whose memory and / or cognitive function has declined due to aging, a patient having amnesia or forgetfulness, a patient with mild cognitive impairment, or a dementia patient.

[13] A method for inhibiting the synapse-eliminating action of amyloid-beta protein in a patient, comprising administering to the patient a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof.

[14] The method according to

[13] , wherein the patient is a dementia patient or a patient having accumulation of Aβ protein in the brain.

[15] The method according to

[14] , wherein the patient is an Alzheimer's disease patient.

[16] A method for inhibiting the production of amyloid-beta protein in a patient, comprising administering to the patient a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof.

[17] The method according to

[16] , wherein the patient is a dementia patient or a patient having accumulation of Aβ protein in the brain.

[18] The method according to

[17] , wherein the dementia patient is an Alzheimer's disease patient.

[19] A method for promoting the clearance of amyloid-beta protein in a patient, comprising administering to the patient a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof.

[20] The method according to

[19] , wherein the patient is a patient with dementia or a patient having accumulation of Aβ protein in the brain.

[21] The method according to

[20] , wherein the patient with dementia is a patient with Alzheimer's disease.

[22] The Crtac1B protein is a polypeptide having an amino acid sequence with 95% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and the C-terminal region fragment is (i) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 7, (ii) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 9, (iii) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 11, or (iv) A polypeptide having 90% or more sequence identity to the protein fragments of (i) to (iii) The method according to any one of [9] to

[21] .

[23] The method according to any one of [9] to

[22] , wherein the cell expressing the Crtac1B protein or a C-terminal region fragment thereof is a neural stem / progenitor cell, an excitatory neuron, or an oligodendrocyte progenitor cell. [Effect of the Invention]

[0014] Since LOTUS / Crtac1B has the effects of reducing Aβ accumulated in the brain, inhibiting the production of Aβ, and suppressing the synaptic reduction effect of Aβ itself, the LOTUS / Crtac1B replacement therapy of the present invention is an epoch-making treatment method that can comprehensively control the effects of Aβ. In addition to the effect of blocking the environment that inhibits nerve regeneration by blocking the receptor on which the nerve regeneration inhibitor acts, LOTUS / Crtac1B also has the effect of promoting neurite outgrowth and synapse formation. Therefore, it is expected that it can act on the remaining neurons not only for mild cognitive impairment or dementia, but also for moderate or more severe cognitive impairment or dementia to improve the symptoms. Not only Alzheimer's type dementia caused by the accumulation of Aβ, but also the decline in memory and cognitive function due to aging can be improved by LOTUS / Crtac1B replacement. Furthermore, it has been confirmed that the memory function is improved by LOTUS / Crtac1B replacement even in healthy young mice. Therefore, it can be effective for various dementias and cognitive impairments including Alzheimer's type dementia, as well as amnesia and amnestic disorder. Since LOTUS / Crtac1B is an endogenous substance, unlike existing antibody drugs for Alzheimer's disease, the risk of side effects is very low. In fact, LOTUS / Crtac1B is abundantly expressed in the brains of healthy people, and no adverse events have been observed even in animal experiments with overexpression of LOTUS.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] The present invention relates to an invention for preventing or treating dementia, enhancing memory and / or cognitive function, inhibiting the synapse-loss effect of Aβ, inhibiting the production of Aβ, or promoting Aβ clearance by administering a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof. As used herein, the term "agent of the present invention" refers to a preventive or therapeutic agent for dementia, an enhancer of memory and / or cognitive function, an agent for inhibiting the synapse-loss effect of Aβ, an Aβ production inhibitor, and an Aβ clearance promoter, which contain a Crtac1B protein or a C-terminal region fragment thereof, a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, or a cell expressing the Crtac1B protein or a C-terminal region fragment thereof as an active ingredient.

[0017] In the present invention, the term "dementia" includes various dementias such as Alzheimer's dementia (also referred to as Alzheimer's disease), vascular dementia (also referred to as cerebrovascular dementia), frontotemporal dementia, dementia due to trauma, Lewy body dementia, and dementia due to Parkinson's disease. Senile dementia is also a form of dementia and includes Alzheimer's dementia and vascular dementia. In addition, the term "Alzheimer's dementia" includes early-onset Alzheimer's disease and late-onset Alzheimer's disease. Early-onset Alzheimer's disease is generally Alzheimer's disease that develops before the age of 65, and late-onset Alzheimer's disease is Alzheimer's disease that develops at the age of 65 or older. Similarly, in dementias other than Alzheimer's dementia, if the dementia patient is under 65 years old, they are referred to as an early-onset dementia patient, and if they are 65 years old or older, they are referred to as a senile dementia patient. When the administration target of the agent of the present invention is a dementia patient, it may be an early-onset dementia patient and a senile dementia patient, or it may be an early-onset dementia patient or a senile dementia patient.

[0018] Various cognitive function scales are used to determine the severity of dementia (mild, moderate, severe). Generally, the evaluation of the degree of cognitive function is mainly conducted using the Clinical Dementia Rating (CDR). In the United States, the MMSE, CDR, and Wechsler Memory Scale-IV Logical Memory II are often used. In the CDR, for each of the six items of memory, orientation, judgment / problem-solving, social adaptation, family situation / interest / concern, and caregiving situation, they are evaluated on a scale of 0 to 3 points based on an evaluation form, and the severity is evaluated by the total score of the six items. Although the definitions may vary from paper to paper, as an example, each item is evaluated as 0: no impairment, 0.5: suspected impairment, 1: mild impairment, 2: moderate impairment, 3: severe impairment. There is a method of evaluating that a total score of 0.5 points for the six items is mild cognitive impairment (MCI), 1 - 6 points is mild dementia, 7 - 12 points is moderate dementia, and 13 - 18 points is severe dementia. In the present invention, the severity of dementia may be evaluated by this method. The dementia targeted in the present invention includes mild dementia, moderate dementia, and severe dementia. When the administration target of the agent of the present invention is a patient with dementia such as Alzheimer's disease, patients with mild, moderate, and severe dementia may be targeted, or patients with a dementia severity of moderate or above (moderate or severe) may be targeted, or severe patients may be targeted.

[0019] The treatment of dementia includes preventing the progression of dementia symptoms and improving dementia symptoms.

[0020] The prevention of dementia includes the treatment of MCI. In one aspect, the agent for preventing or treating dementia is an agent for treating MCI or dementia. Generally, when the cognitive function is somewhat decreased but not at a level that interferes with daily life, it is diagnosed as MCI, and when it is at a level that interferes with daily life, it is diagnosed as dementia. By administering the dementia treatment agent of the present invention at the stage of MCI, the progression to dementia can be prevented, and improvement of MCI can also be expected. MCI patients include elderly MCI patients and young MCI patients. In the present invention, MCI patients aged 65 years or older are referred to as elderly MCI patients, and MCI patients under 65 years old are referred to as young MCI patients. When the subject of administration of the agent of the present invention is an MCI patient, it may be elderly and young MCI patients, elderly MCI patients, or young MCI patients.

[0021] In the present invention, amnesia includes various amnesias such as senile amnesia, anterograde amnesia, retrograde amnesia, transient amnesia, dissociative amnesia, etc. Further, forgetfulness is a memory disorder that does not interfere with daily life and is not diagnosed as amnesia, and includes a memory disorder at a level diagnosed as suspected amnesia.

[0022] The patients targeted by the present invention include dementia patients including the above specific examples, patients with amnesia or forgetfulness including the above specific examples, the elderly (the elderly with amnesia or forgetfulness) whose memory and / or cognitive function has decreased due to aging, and patients with accumulation of Aβ protein in the brain. The patient is typically a human. The elderly are humans aged 65 years or older, and may be, for example, 70 years or older, 75 years or older, or 80 years or older.

[0023] Patients with accumulation of Aβ protein in the brain include not only patients presenting with decline in memory and / or cognitive function, but also patients without decline in memory and / or cognitive function. The main example of a disease or condition in which Aβ accumulates in the brain is Alzheimer's disease, but the patients with Aβ accumulation in the brain targeted by the present invention are not limited to Alzheimer's disease, and widely include patients in whom Aβ accumulation in the brain has been confirmed by amyloid PET, cerebrospinal fluid (CSF) examination, or equivalent diagnostic methods, etc.

[0024] Patients or subjects in need of enhancement of memory and / or cognitive function include the elderly whose memory and / or cognitive function has declined due to aging, patients with amnesia or forgetfulness (an example of patients or subjects in need of enhancement of memory function), and patients with dementia. In one aspect, the patients or subjects in need of enhancement of memory and / or cognitive function are the elderly whose memory and / or cognitive function has declined due to aging, including patients with senile forgetfulness. In one aspect, the patients or subjects in need of enhancement of memory and / or cognitive function are patients with dementia. In one aspect, the patients or subjects in need of enhancement of memory and / or cognitive function are patients with senile dementia. Senile dementia includes Alzheimer's type dementia and vascular dementia. Also, in one aspect, the patients in need of enhancement of memory and / or cognitive function are patients with Alzheimer's type dementia, including patients with cognitive impairment due to early-onset Alzheimer's disease and patients with cognitive impairment due to late-onset Alzheimer's disease. Early-onset Alzheimer's disease generally refers to Alzheimer's disease that develops before the age of 65.

[0025] Agents that inhibit the synapse-loss effect of Aβ protein, Aβ protein production inhibitors, and Aβ protein clearance promoters can be preferably administered to patients in whom Aβ protein accumulation in the brain has been confirmed. Accumulation of Aβ in the brain can be examined, for example, by amyloid PET, cerebrospinal fluid (CSF) examination, or equivalent diagnostic methods. When findings suggesting Aβ pathology are confirmed by these diagnostic methods, administration of an agent that inhibits the synapse-loss effect of Aβ protein, an Aβ protein production inhibitor, and an Aβ protein clearance promoter can improve the decline in memory and cognitive function and the symptoms based thereon, or suppress its progression. A preventive or therapeutic agent for dementia and an enhancer of memory and / or cognitive function may also have patients with confirmed Aβ protein accumulation in the brain as the administration target.

[0026] The substance used as an active ingredient in the agent of the present invention (i.e., a preventive or therapeutic agent for dementia, an enhancer of memory and / or cognitive function, an agent that inhibits the synapse-loss effect of Aβ, an Aβ production inhibitor, and an Aβ clearance promoter) is any of the following. Two or more of (1) to (3) may be used in combination. For example, an agent of the present invention containing (1) as an active ingredient and an agent of the present invention containing (2) as an active ingredient may be administered to a patient in combination. (1) Crtac1B protein or a C-terminal region fragment thereof. (2) A vector capable of expressing Crtac1B protein or a C-terminal region fragment thereof. (3) Cells that express Crtac1B protein or a C-terminal region fragment thereof.

[0027] The Crtac1B protein itself is known, and the mRNA sequence of the Crtac1B gene and the amino acid sequence of the Crtac1B protein encoded thereby are registered in NCBI's GenBank under NM_001206528.3. These sequences are shown as SEQ ID NOs: 1 and 2 in the sequence listing. SEQ ID NO: 3 shows the sequence of the coding region in the nucleotide sequence shown in SEQ ID NO: 1.

[0028] As the Crtac1B protein, a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 can preferably be used. Also, when there are mutations such as substitutions in some amino acids, if it is a conservative substitution, a mutation in a site that is not important for activity, or when the sequence identity is sufficiently high, it is well-known that the properties and activity of the original protein are not impaired. Therefore, a polypeptide having an amino acid sequence that is partially different from the amino acid sequence of SEQ ID NO: 2 and has an identity of 95% or more, or 98% or more with SEQ ID NO: 2 can also maintain the antagonistic action against NgR1 and PirB possessed by the Crtac1B protein having the amino acid sequence shown in SEQ ID NO: 2, and furthermore, it is highly probable that the binding property to BACE1 is also maintained. Thus, it can be used as the Crtac1B protein which is the active ingredient of the agent of the present invention.

[0029] Conservative substitutions, that is, substitutions to amino acids with similar chemical properties, are highly likely not to impair the properties and activity of the protein. Amino acids with similar side chains have similar chemical properties. When classifying amino acids by the similarity of their side chains, for example, it can be classified into groups such as amino acids having aliphatic side chains (glycine, alanine, valine, leucine, isoleucine), amino acids having aliphatic hydroxyl side chains (serine, threonine), amino acids having amide-containing side chains (asparagine, glutamine), amino acids having aromatic side chains (phenylalanine, tyrosine, tryptophan), amino acids having basic side chains (arginine, lysine, histidine), amino acids having acidic side chains (aspartic acid, glutamic acid), amino acids having sulfur-containing side chains (cysteine, methionine), and the like. Substitutions to another amino acid belonging to the same group are conservative substitutions.

[0030] The identity of amino acid sequences refers to the percentage obtained by aligning two amino acid sequences to be compared so that as many amino acid residues as possible match, and then dividing the number of matching amino acid residues by the total number of amino acid residues. When performing the above alignment, gaps are appropriately inserted into one or both of the two sequences to be compared as necessary. Such alignment of sequences can be performed using well-known programs such as BLAST, FASTA, CLUSTAL W, etc. When gaps are inserted, the above total number of amino acid residues is the number of residues counted with one gap counted as one amino acid residue. When the total number of amino acid residues counted in this way is different between the two sequences to be compared, the identity (%) is calculated by dividing the total number of amino acid residues in the longer sequence by the number of matching amino acid residues.

[0031] In one aspect, the active ingredient of the agent of the present invention is the Crtac1B protein, a vector capable of expressing the Crtac1B protein, or a cell expressing the Crtac1B protein.

[0032] The antagonistic effect of Crtac1B against NgR1 is known to exist in the region of residues 446 to 646 in the Crtac1B protein sequence of SEQ ID NO: 2 (Kurihara, Y., et al., Biochem. Biophys. Res. Commun. 2012, 418, 390 - 395.). This region is a region containing the UnbV_ASPIC (UA) domain (residues 460 to 518) and the EGF_CA (EC) domain (residues 559 to 605), and it is also known that fragments containing only the UA domain or only the EC domain can bind to NgR1 (Kurihara, Y., et al. 2012 cited above). The nucleotide sequence encoding the region of residues 446 to 646 (also referred to as the UA / EC domain) and its amino acid sequence are shown in SEQ ID NO: 6 and 7, the nucleotide sequence encoding the UA domain and the amino acid sequence of the UA domain are shown in SEQ ID NO: 8 and 9, and the nucleotide sequence encoding the EC domain and the amino acid sequence of the EC domain are shown in SEQ ID NO: 10 and 11, respectively. Therefore, the C-terminal region fragment of Crtac1B, specifically, (i) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 7, (ii) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 9, (iii) A Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 11, or (iv) A polypeptide having 90% or more, such as 95% or more, 97% or more, or 98% or more sequence identity with the protein fragments of (i) to (iii) also maintains the binding ability to NgR1 and PirB, and is considered to be able to exert the effects of preventing and treating dementia, enhancing cognitive and / or memory functions, suppressing synapse loss caused by Aβ, inhibiting Aβ production, and promoting Aβ clearance, similar to the full-length Crtac1B protein. Therefore, it can be used as an active ingredient of the agent of the present invention.

[0033] In one embodiment, the C-terminal region fragment of Crtac1B is (i) a Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 7, (ii) a Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 9, or (iii) a Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 11. In another embodiment, the C-terminal region fragment of Crtac1B is a polypeptide of the amino acid sequence shown in SEQ ID NO: 7, 9 or 11, or a polypeptide having 90% or more, such as 95% or more, 97% or more, or 98% or more sequence identity with the polypeptide. In another embodiment, the C-terminal region fragment of Crtac1B is a polypeptide of the amino acid sequence shown in SEQ ID NO: 7, 9 or 11. In another embodiment, the C-terminal region fragment of Crtac1B is a Crtac1B protein fragment containing the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide having 90% or more, such as 95% or more, 97% or more, or 98% or more sequence identity with the fragment. In another embodiment, the C-terminal region fragment of Crtac1B is a polypeptide of the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide having 90% or more, such as 95% or more, 97% or more, or 98% or more sequence identity with the polypeptide.

[0034] An agent containing the Crtac1B protein of (1) or a fragment of its C-terminal region as an active ingredient is a protein preparation. The Crtac1B protein or a fragment of its C-terminal region can be prepared by known genetic engineering techniques. For example, the DNA encoding the Crtac1B protein can be prepared using a human cDNA library or cDNA prepared from mRNA extracted from human cultured nerve cells as a template, and performing PCR using a pair of primers designed to amplify the nucleotide sequence encoding the full-length Crtac1B protein shown in SEQ ID NO: 1. The C-terminal region fragment of the Crtac1B protein can be obtained by performing PCR using a pair of primers designed to amplify the region encoding the C-terminal region fragment among the nucleotide sequence of SEQ ID NO: 1. The cDNA encoding the Crtac1B protein or a fragment of its C-terminal region containing mutations such as amino acid substitutions can be prepared by introducing mutations into the PCR-amplified cDNA fragment by conventional methods. By incorporating the prepared cDNA into an appropriate expression vector and introducing it into a host cell, and expressing the polypeptide in the host cell, the desired Crtac1B protein or a fragment of its C-terminal region can be obtained. Various expression vectors and host cells are known and many commercial products are available, so they are easily obtainable.

[0035] Isolation and purification of the target polypeptide from the host cell can be carried out by combining known separation operations. For example, treatment with a denaturing agent such as urea or a surfactant, sonication, enzymatic digestion, salting out or solvent fractionation precipitation method, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing electrophoresis, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, etc. can be mentioned, but it is not limited to these.

[0036] (1)'s Crtac1B protein or its C-terminal region fragment may be one to which techniques such as glycosylation, PEG addition, or using at least some of the amino acids constituting the polypeptide as D-amino acids, which are techniques carried out for improving in vivo stability and the like in peptide pharmaceuticals, are applied. Such techniques are well-known as described, for example, in J Am Chem Soc. 2004 Nov 3;126(43):14013-22 and Angew Chem Int Ed Engl. 2004 Mar 12;43(12):1516-20 (glycosylation), Clin Nephrol. 2006 Mar;65(3):180-90. and Proc Natl Acad Sci USA. 2005 Sep 6;102(36):12962-7. (PEG addition), J Pharmacol Exp Ther. 2004 Jun;309(3):1190-7 and J Pharmacol Exp Ther. 2004 Jun;309(3):1183-9. (utilization of D-amino acids), etc., and have already been used in the field of peptide pharmaceuticals.

[0037] Alternatively, when a recombinant polypeptide is produced by genetic engineering techniques, it can undergo various post-translational modifications (removal of N-terminal methionine, N-terminal acetylation, glycosylation, limited proteolysis by intracellular proteases, myristoylation, isoprenylation, phosphorylation, etc.) in host cells. However, as long as such post-translationally modified forms of the polypeptide also have an antagonistic effect against NgR1 and PirB, and can exert an effect of preventing and treating dementia, enhancing cognitive and / or memory functions, suppressing synapse loss caused by Aβ, inhibiting Aβ production, and promoting Aβ clearance, they can be used as an active ingredient of the agent of the present invention and are included in the scope of the present invention.

[0038] In addition, for the convenience of polypeptide production and other reasons, polypeptides with an arbitrary amino acid sequence added, such as polypeptides with a Flag tag, His tag, GST added, or polypeptides in a form fused with other proteins or fragments thereof, also utilize the present invention because the region that exhibits the various actions described above as the agent of the present invention is the region of the Crtac1B protein or a fragment of its C-terminal region. Therefore, such fusion proteins are also included in the scope of the present invention. In the case of an amino acid sequence fused with other proteins, etc., the amino acid sequence identity shall be calculated by extracting only the region corresponding to the Crtac1B protein or a fragment of its C-terminal region. For example, in the case of a polypeptide with a His tag added, the identity is calculated between the regions excluding the His tag.

[0039] The protein preparation according to the present invention may consist only of a polypeptide, but usually, it is formulated by appropriately mixing additives such as pharmacologically acceptable carriers, diluents, excipients, etc., suitable for each dosage form. Formulation methods and usable additives are well-known in the field of pharmaceutical preparations, and any method and additive can be used. Specific examples of additives include physiological buffer solutions, diluents such as trometamol; stabilizers such as polyoxyethylene polyoxypropylene glycol; excipients such as sugar, lactose, corn starch, calcium phosphate, sorbitol, glycine, etc.; binders such as syrup, gelatin, gum arabic, sorbitol, polyvinyl chloride, tragacanth, etc.; lubricants such as magnesium stearate, polyethylene glycol, talc, silica, etc., but are not limited thereto. As the administration route of the protein preparation according to the present invention, intrathecal administration, intraventricular administration, or intracerebral administration is preferred, but it can also be used for other parenteral administrations such as intravenous administration, intramuscular administration, subcutaneous administration, intraarterial administration, or oral administration.

[0040] The dosage of the protein preparation according to the present invention may be any amount as long as the intended effect can be obtained, and it is appropriately selected according to the patient's symptoms, severity, age, weight, etc. Usually, the amount of the active ingredient per day for a patient is 0.001 mg to 1000 mg, for example, 0.01 mg to 100 mg per kg of body weight, and it can be administered once or divided into several times. The administration may be daily or once every one to several days.

[0041] (2) An agent containing as an active ingredient a vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof is a gene therapy agent. The vector is not particularly limited as long as it can be expressed in the cells of the patient to be administered (typically in human cells, particularly in human nerve cells), and it may be a plasmid vector or a viral vector, and any vector known in the field of gene therapy agents may be used. Preferred examples of the viral vector include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, etc. Among them, in particular, an adeno-associated viral vector with high central migration ability (such as AAV9 type) can be preferably used. In one embodiment, the agent of the present invention (that is, a preventive or therapeutic agent for dementia, an enhancer of memory and / or cognitive function, an agent that inhibits the synapse loss action of Aβ, an Aβ production inhibitor, and an Aβ clearance promoter) may be an agent containing as an active ingredient an adeno-associated viral vector capable of expressing the Crtac1B protein or a C-terminal region fragment thereof, for example, an adeno-associated viral vector of type 9 (AAV9 type) capable of expressing the Crtac1B protein or a C-terminal region fragment thereof. The adeno-associated viral vector of type 9 capable of expressing the Crtac1B protein or a C-terminal region fragment thereof may be, for example, a recombinant adeno-associated virus having a capsid of type AAV9 and capable of expressing the Crtac1B protein or a C-terminal region fragment thereof.

[0042] A polynucleotide (DNA or RNA) encoding the Crtac1B protein or a C-terminal region fragment thereof can be prepared as described above. The "Crtac1B protein" and the "C-terminal region fragment of Crtac1B" are as defined above. The polynucleotide encoding the Crtac1B protein can be, for example, a polynucleotide having a nucleotide sequence with 90% or more, such as 95% or more, or 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 3. When the polynucleotide encoding the C-terminal region fragment of Crtac1B is a polypeptide (UA / EC domain fragment) with the amino acid sequence shown in SEQ ID NO: 7, it can be a polynucleotide having a nucleotide sequence with 90% or more, such as 95% or more, or 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 6. When the C-terminal region fragment is a polypeptide (UA domain fragment) with the amino acid sequence shown in SEQ ID NO: 9, it can be a polynucleotide having a nucleotide sequence with 90% or more, such as 95% or more, or 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 8. When the C-terminal region fragment is a polypeptide (EC domain fragment) with the amino acid sequence shown in SEQ ID NO: 11, it can be a polynucleotide having a nucleotide sequence with 90% or more, such as 95% or more, or 98% or more identity to the nucleotide sequence shown in SEQ ID NO: 10. The incorporation of the polynucleotide encoding the Crtac1B protein or its C-terminal region fragment into a vector can be carried out by methods well known to those skilled in the art.

[0043] The gene therapy agent according to the present invention may consist only of the vector in (2), but usually, additives such as pharmacologically acceptable carriers, diluents, excipients, etc., suitable for each dosage form are appropriately mixed and formulated. Specific examples of the additives are as described above. The administration route is preferably intrathecal administration, intraventricular administration, or intracerebral parenchymal administration, but it can also be used for other parenteral administrations such as intravenous administration, intramuscular administration, subcutaneous administration, and intraarterial administration.

[0044] The dosage of the gene therapy agent according to the present invention may be any amount as long as the intended effect can be obtained, and it is appropriately selected according to the patient's symptoms, severity, age, weight, etc. Usually, the amount of the active ingredient per day for the patient is about 0.1 μg to 100 mg, for example, about 1 μg to 10 mg per kg of body weight. Although gene therapy agents are generally used in single-dose administration, the number of administrations in the case where the agent of the present invention is a gene therapy agent is not limited to single-dose administration, and it may be single-dose administration or multiple (2, 3, or 4 or more) administrations.

[0045] An agent containing, as an active ingredient, cells expressing the Crtac1B protein of (3) or a C-terminal region fragment thereof can also be referred to as an ex vivo gene therapy agent. As the cells, neural stem / progenitor cells, excitatory neurons (neurons using glutamate as a neurotransmitter), or oligodendrocyte progenitor cells can preferably be used. These cells can be prepared by inducing differentiation from pluripotent stem cells such as induced pluripotent stem (iPS) cells and mesenchymal stem cells. When the patient is human, human-derived cells are used as the cells expressing the Crtac1B protein or a C-terminal region fragment thereof. Methods for producing iPS cells are well known. Mesenchymal stem cells can be collected from umbilical cord blood.

[0046] Neural stem / progenitor cells are a cell population containing neural stem cells and neural progenitor cells, and can be prepared from iPS cells by the method described in, for example, Itakura G et al., 2015, PLoS One 10, e0116413. Specifically, iPS cells are cultured in a standard ES cell medium together with irradiated mouse embryonic fibroblasts using a gelatin-coated (0.1%) culture dish to form embryoid bodies (EBs). 30 days after EB formation, the EBs are enzymatically treated to be separated into single cells, and the cells are cultured in suspension for 12 days in a serum-free medium containing EGF and bFGF to form neurospheres (spherical cell masses). By subculturing this primary iPS cell-derived neurosphere 2 to 3 times, iPS cell-derived neural stem / progenitor cells can be obtained.

[0047] Excitatory neurons can be prepared by culturing neural stem / progenitor cells in an appropriate serum-free medium in the presence of differentiation-inducing factors for neurons such as retinoic acid and neural cell growth factors such as b-FGF to induce differentiation. The specific procedure for preparing excitatory neurons from pluripotent stem cells is as described in Preparation Example 1 of the following Examples.

[0048] Introduction of a polynucleotide encoding the Crtac1B protein or a C-terminal region fragment thereof may be performed on iPS cells, or on single cells separated by enzymatic treatment of primary or passage 2 or 3 neurospheres, or on neural stem / progenitor cells after induction of differentiation. When using excitatory neurons, in addition to the above, it may also be performed on excitatory neurons after induction of differentiation into neurons. When introducing a polynucleotide into neurosphere cells, the cells after introduction can be cultured in suspension to form neurospheres again and passaged. Introduction of the polynucleotide into cells can be carried out, for example, by infecting the cells with a recombinant virus in which a polynucleotide encoding the Crtac1B protein or a C-terminal region fragment thereof is incorporated into an adeno-associated virus or a lentiviral vector. Note that iPS cell-derived neural stem / progenitor cells expressing LOTUS / Crtac1B themselves are known and described in Ito et al., Stem Cell Reports 2021, 16:1-15.

[0049] Oligodendrocyte progenitor cells can be prepared from pluripotent stem cells, for example, by the method described in Douvaras and Fossati, Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells. Nat Protoc 10, 1143-1154 (2015). The specific procedure is as described in Preparation Example 2 of the following Examples.

[0050] The ex vivo gene therapy agent according to the present invention is preferably used by intrathecal administration, intraventricular administration, or intracerebral parenchymal administration in the same manner as protein preparations and gene therapy agents. However, parenteral administrations such as intravenous administration and intraperitoneal administration are also possible, and it can be formulated as an intravenous drip agent, drip agent, or injection containing the cells of (3) in a medium suitable for these administration routes. As the medium, for example, an aqueous medium (e.g., high-purity sterilized water) containing about 2.5 to 5% human serum albumin, about 0.2 to 0.5% sodium chloride, and about 3 to 8% dimethyl sulfoxide as a cryoprotectant can be used. The number of cells of (3) in the preparation for one administration (e.g., one bag of intravenous drip agent) can be appropriately set according to the age, weight, symptoms, etc. of the patient. For example, the number of viable cells can be about 1 x 10 5 ~1 x 10 9 cells. The ex vivo gene therapy agent is generally used for single administration. However, when the agent of the present invention is an ex vivo gene therapy agent, the number of administrations is not limited to single administration, and it may be single administration or multiple administrations (2 times, 3 times, or 4 times or more).

[0051] The agent of the present invention is usually provided with an attached document describing usage, dosage, etc. enclosed or attached. In other words, in the production of the agent of the present invention, Crtac1B protein or its C-terminal region fragment, a vector capable of expressing Crtac1B protein or its C-terminal region fragment, or cells expressing Crtac1B protein or its C-terminal region fragment, and an attached document can be used. For example, the agent of the present invention may include an attached document in its package. The attached document may describe that the patient to whom the agent is administered is a patient with moderate or more severe dementia, for example, a patient with moderate or more severe Alzheimer's dementia.

Example

[0052] Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

[0053] Example 1: BACE1 Experiment [Materials and Methods] Construction of expression plasmids Human BACE1 (NM_012104.4, SEQ ID NO: 12) or human APP695 (NM_201414.3, SEQ ID NO: 14) coding sequences were incorporated into the vector pcDNA3.1(+) vectors (Invitrogen) to generate the human BACE1 / pcDNA3.1+ plasmid and the human APP / pcDNA3.1+ plasmid. Using the human cDNA library brain (TAKARA) as a template, PCR was performed under the conditions described in Table 2 using the enzyme KOD plus (TOYOBO) and the primers shown in Table 1 to amplify the coding sequences of human BACE1 and human APP695. The PCR products and the vector were treated with restriction enzymes, and the PCR products were incorporated into the vector using the Ligation-Convenience Kit (NIPPON GENE CO, LTD.). The plasmids produced were subjected to sequence analysis (Fasmac).

[0054] [Table 1]

[0055] [Table 2]

[0056] Preparation of soluble LOTUS HEK293T cells were seeded at 9.0×10 in a 15 cm culture dish 6Cells / dish were seeded with a total volume of 30 ml of medium. After culturing for 2 days (37°C, 5% CO2), the plasmid was introduced with 0.1% PEI (Polyethyleneimine), and then cultured for 4 days. As the plasmid, one in which LOTUS (SEQ ID NO: 4) without a membrane-binding region was incorporated into a vector having the sequence of SBP-hFc (Human IgG1e3) tag was used. The target protein (SBP-hFc-human LOTUS) was secreted into the culture supernatant. After culturing, the supernatant was collected and completely separated from the supernatant using an ultracentrifuge (40,000 rpm, 4°C, 1 hour). The centrifuged supernatant was passed through High Capacity Streptavidin Agarose Resin (Thermo Scientific) that specifically binds to SBP (4°C, 10 sec / drop, Overnight). Then, the target protein was competitively eluted with a Biotin solution (2 mM Biotin / PBS) that binds more strongly than Streptavidin and collected in a 0.6 ml siliconized tube. The recovered protein (SBP-hFc-human LOTUS) was subjected to CBB staining after SDS-PAGE, and the concentration was calculated based on a BSA sample with a known concentration diluted for the calibration curve.

[0057] Pull-down assay COS-7 cells were seeded in a 6 cm cell culture plate (Greiner bio-one) at 5.0×10 5Cells / well were seeded with a total volume of 2 ml of medium. After incubation for 4 hours (37 °C, 5% CO2), SBP-FLAG-human LOTUS (a 141 bp nucleotide sequence encoding SBP-FLAG (SEQ ID NO: 27) (SEQ ID NO: 26) ligated to a 1938 bp nucleotide sequence encoding human LOTUS (SEQ ID NO: 2) (SEQ ID NO: 3); SEQ ID NOs: 28, 29) and a plasmid encoding human BACE1 or human APP were introduced using Lipofectamine 3000. After incubation for 2 days (37 °C, 5% CO2), the cells were transferred onto ice and washed twice with cold PBS. Proteins were recovered using lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich), 10 μg / ml leupeptin (Nacalai Tesque), 0.1 U / ml aprotinin (Sigma-Aldrich), 50 μM p-amidinophenyl-methylsulfonyl fluoride (Nacalai Tesque)). The recovered cell lysate was stirred (4 °C, 10 min, mild rotation) and then centrifuged (15,000 rpm, 4 °C, 10 min). The supernatant was recovered and 15 μl / sample of SBP-specific beads (Streptavidin agarose: GE Healthcare) was added and mixed (4 °C, 3.5 h, mild rotation). Thereafter, the beads were washed with 1% Tryton-X / MilliQ and centrifuged (6,400 g, 4 °C, 10 min), and this operation was repeated 4 times. Reduction treatment was performed at 100 °C for 7 min with 2.5% mercaptoethanol / 1×sample buffer, and analysis was performed by Western blot. The pull-down assay for soluble LOTUS was performed by the following two methods. (i) The purified SBP-hFc-human LOTUS or SBP-hFc (Tag) was diluted to a final concentration of 1 μM in the maintenance medium and added to COS-7 cells that were forced to express BACE1 or APP. Incubate for 30 minutes (37 °C, 5% CO2), and the protein was recovered using lysis buffer. (ii) The purified SBP-hFc-human LOTUS or SBP-hFc (Tag) was added to the cell lysate of COS-7 cells that were forced to express BACE1 at a final concentration of 1 μM and mixed (4 °C, overnight, mild rotation).

[0058] (i) and (ii) The cell lysates obtained were mixed with 15 μl / sample of Streptavidin agarose resin (4 °C, 4 h, mild rotation). Then, the operation of washing the beads with 1% Tryton-X / MilliQ and centrifuging (6,400 g, 4 °C, 10 min) was repeated 4 times. It was reductively treated at 100 °C for 7 min with 2.5% mercaptoethanol / 1×sample buffer and analyzed by Western blot.

[0059] Western blot analysis The extracted protein was reductively treated at 100 °C for 7 min with 2.5% mercaptoethanol / 1× sample buffer. It was electrophoresed on an 8-12% polyacrylamide gel (10 mA / gel, 10 min → 20 mA / gel, 90 min) and transferred to a PVDF membrane (Millipore) (100 mA, 60 min). Blocking was performed with 5% skim milk / TBST for BACE1, SBP, and BACE1, and 5% BSA / TBST for β-Actin, sAPPβ, and β-CTF (room temperature, 60 min, shaking). Next, a primary antibody reaction (4 °C, overnight, shaking) was carried out using a BACE1 antibody (MAB5308, Merck Millipore, 1 / 5000, 5% skim milk / TBST), an SBP antibody (sc-101595, Santa Cruz Biotechnology, 1 / 10000, 5% BSA / TBST), a β-CTF antibody (A8717, R&D SYSTEMS, 1 / 5000, 5% BSA / TBST), an sAPPβ antibody (IL-902, IBL, 1 / 500, 5% BSA / TBST), and a β-Actin antibody (AC04, Wako, 1 / 10000, 5% BSA / TBST). Secondary antibodies were reacted using Peroxidase-conjugated AffiniPure Donkey anti-mouse IgG (715-035-020: Jackson ImmunoReseach LABORATORIES, INC) and Peroxidase-conjugated AffiniPure Goat anti-rabbit IgG (111-035-003: Jackson ImmunoReseach LABORATORIES, INC) (room temperature, 1 h, shaking). Protein detection was performed using a chemiluminescence reagent (GE healthcare, Millipore, TAKARA) as a substrate and photographed with a LAS-4000 (Fuji Film) image analyzer.

[0060] Quantification of APP Fragment Proteins COS-7 cells were seeded in a 6 cm cell culture plate (Greiner bio-one) at 5.0×10 5 cells / well with a total medium volume of 2 ml. After incubation for 4 hours (37 °C, 5% CO2), plasmids encoding human BACE1, human APP, and human LOTUS were introduced using Lipofectamine 3000 and incubated for 2 days (37 °C, 5% CO2). The cells were transferred to ice, and the collected culture supernatant was centrifuged (800 g, 10 min, 4 °C) and reductively treated at 100 °C for 7 min with 2.5% mercaptoethanol / 1× sample buffer. The cell lysate was obtained by washing the cells twice with PBS (4 °C) and then collecting them using lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM sodium orthovanadate (Sigma-Aldrich), 0.1 U / ml aprotinin (Sigma-Aldrich), 50 μM p-amidinophenyl-methylsulfonyl fluoride (Nacalai Tesque)). The cell lysate was centrifuged (15,000 rpm, 10 min, 4 °C), and the supernatant was collected. It was reductively treated at 100 °C for 7 min with 2.5% mercaptoethanol / 1× sample buffer and analyzed by Western blot. The results were statistically processed by the unpaired t-test method with a significance level (p-value) of 5%.

[0061] BACE1 Assay The activity of BACE1 was measured using the Sensolyte 520 β-secretase assay kit (Anaspec). Fluorescent dye molecules of donor and acceptor are included at both ends of the APP peptide (substrate) containing the Swedish mutation, and fluorescence that can be measured is emitted upon BACE1 cleavage. Fluorescence was measured according to the protocol of the kit. To 40 μl of BACE1 recombinant protein diluted 50-fold with 1x Assay buffer (pH 4.5), 10 μl each of 2.5 μM SBP-hFc-LOTUS or 2.5 μM SBP-hFc (Tag) and 5 μM LY2886721 (BACE1 inhibitor) were added and reacted (10 min, 37 °C). Then, 50 μl of Substrate diluted 100-fold with 1x Assay buffer (pH 4.5) was added and reacted (30 min, 37 °C). The fluorescence wavelength at an excitation wavelength / fluorescence wavelength of 490 nm / 520 nm was measured using Varioskan (Thermo). The results were statistically analyzed by the One-Way ANOVA Dunnett method, with a significance level (risk rate) of 5%.

[0062] [Results] LOTUS binds to BACE1 BACE1 undergoes post-translational modification of palmitoylation and localizes to membrane microdomains "rafts" approximately 10 nm to 200 nm in size formed from cholesterol and sphingolipids (Kulandaivelu S., Gopal T., Biochim Biophys Acta, 2010; 1801(8): 860-867). Since LOTUS is a GPI-anchored protein, it was considered possible that LOTUS and BACE1 localize and bind in a proximal environment within the raft. Also, there are a membrane-bound type (SEQ ID NO: 2) and a soluble type (SEQ ID NO: 5) in which the C-terminus is cleaved and secreted for LOTUS. Since the mechanisms of action of membrane-bound LOTUS and soluble LOTUS on NgR1 are different, it was considered possible that the binding modes to BACE1 are different between the membrane-bound and soluble types. Therefore, the binding of both membrane-bound and soluble LOTUS to BACE1 was examined.

[0063] First, to examine membrane-bound LOTUS, COS-7 cells were co-expressed with human BACE1 and SBP-FLAG-LOTUS and subjected to a pull-down assay. As a result, BACE1 was detected by an anti-BACE1 antibody in the lysate pulled down by SBP (Figure 1A). Next, to examine soluble LOTUS, purified SBP-hFc-LOTUS or SBP-hFc (Tag) was added at 1 μM to COS-7 cells overexpressing human BACE1. As a result of the pull-down assay, BACE1 was not detected in the lysate pulled down by SBP (Figure 1B). On the other hand, when SBP-hFc-LOTUS or SBP-hFc (Tag) was added at 1 μM to the cell lysate of COS-7 cells overexpressing human BACE1, BACE1 was detected in the lysate pulled down by SBP (Figure 1C). From the above, it was found that both membrane-bound and soluble LOTUS bind to BACE1.

[0064] Membrane-bound LOTUS inhibits APP cleavage by BACE1 To examine whether LOTUS is involved in the Aβ production process, human LOTUS, human BACE1, and human APP were co-expressed in COS-7 cells, and the expression levels of β-CTF and sAPPβ produced by BACE1 cleavage of APP were quantified. It was confirmed that there was no difference in the BACE1 expression level between the cells expressing LOTUS and the control (mock) (Figures 2A, 2B). β-CTF is a membrane-bound C-terminal fragment produced by APP upon BACE1 cleavage. In the cells expressing LOTUS, the expression level of β-CTF was significantly decreased compared to the control (mock) (Figures 2C, 2D). On the other hand, sAPPβ is the extracellular region secreted by APP upon BACE1 cleavage. Therefore, sAPPβ in the culture supernatant was quantified. In the cells expressing LOTUS, the expression level of sAPPβ was significantly decreased compared to the control (mock) (Figures 2E, 2F).

[0065] Soluble LOTUS inhibits BACE1 activity To examine whether LOTUS directly controls the activity of BACE1, BACE1 activity was measured using a β-secretase assay kit (Anaspec). LY2886721 (BACE inhibitor), which has been shown to inhibit the activity of BACE1, was used as a control. Purified 2.5 μM SBP-hFc-LOTUS or 2.5 μM SBP-hFc (Tag), 5 μM inhibitor, and 2 mM biotin / PBS (vehicle control) were each reacted with BACE1 recombinant protein for 10 minutes. Subsequently, the substrate attached to the kit (FRET peptide of APP with Swedish mutation) was added and reacted at 37 °C under a pH 4.5 environment for 30 minutes. BACE1 activity can be quantified by measuring the fluorescence wavelength at an excitation wavelength / fluorescence wavelength of 490 nm / 520 nm. Soluble LOTUS significantly reduced BACE1 activity compared to the control (Tag) (Figure 3).

[0066] Example 2: Microglia Experiment [Materials and Methods] Primary microglia cells The cortices of C57B6J mice at postnatal days 0 - 3 were excised, dispersed with 0.125% trypsin (37 °C, 5 minutes), and then suspended in DMEM (10% heat-inactivated FBS + 0.5% Penicillin streptomycin) + 0.01 mg / ml DNaseI medium. Subsequently, after filtering the cells through a 70 μm filter, approximately 4.5×10 6 cells were seeded in a T75 flask coated with 10 μg / ml PLL (Polyethylenimin), and the mixed glial cells were cultured for 14 days (37 °C, 5% CO2). After 14 days, only microglia were detached from the mixed glial cells, dispersed again with DMEM (high glucose), and seeded.

[0067] Preparation of Aβ After dissolving Biotin-LC-Aβ(1-42) in 1% aqueous ammonia, it was diluted with PBS to a final concentration of 100 μM. Then, it was polymerized by incubating at 37 °C for 48 hours (oligomer Aβ: hereinafter abbreviated as Aβ). In the phagocytosis assay, after mixing with Avidin-pHrodo in equimolar amounts and allowing to stand on ice for 30 minutes, an oligomer Aβ-pHrodo complex (Aβ-pHrodo) was prepared.

[0068] ELISA, WST assay Primary microglial cells were treated with purified proteins [SBP-FLAG (Tag), SBP-FLAG-soluble LOTUS (sLOTUS)] (final concentrations of 200, 500 nM) and Aβ (final concentration of 1 μM) for 24 hours, and the cytokines contained in the supernatant were measured according to an ELISA Kit (Invitrogen). On the other hand, a WST kit (Dojin) was added to the cell bodies so that DMEM (high glucose):WST = 100:10, and the absorbance at 450 nm was measured after 4 hours.

[0069] Phagocytosis assay Microglial cultured cells were treated with purified proteins [SBP-FLAG (Tag), SBP-FLAG-soluble LOTUS (sLOTUS)] (final concentration of 500 nM) and Aβ-pHrodo (500 ng) for 4 hours, and then fixed with 4% PFA / PBS (room temperature, 30 minutes, light-shielded). After permeabilization with 0.3% TritonX-100 / PBS (room temperature, 10 minutes), it was reacted with DAPI / PBS (1 / 2000) for 10 minutes and then observed with a Nikon fluorescence microscope. The number of cells (Aβ-pHrodo / DAPI) was counted using ImageJ.

[0070] [Results] The survival rate of microglia is improved by sLOTUS Microglia are stably maintained by repeating proliferation and apoptosis from birth to old age (Katharina Borst et al., Immunity, 2021 Oct 12;54(10):2194-2208.). In chronic neuropathy and aging, it has been reported that microglial dysfunction contributes to the progression of the disease state (Yanling Hu et al., Cell Rep. 2021 Jun 8;35(10):109228. and T Kreisel et al., Mol Psychiatry. 2014 Jun;19(6):699-709.). Therefore, the relationship between LOTUS and the viability of microglia was examined. sLOTUS was added to primary microglial cells, and the number of living cells was measured using the WST assay. Since the WST assay is a color reaction using the reduction reaction by mitochondrial dehydrogenase in living cells, it can be seen that the higher the absorbance, the more living cells there are. As a result, it was revealed that when treated with sLOTUS for 24 hours compared to the control with the purified Tag of sLOTUS, the number of living microglia increased by about 40% (Figure 4A). Furthermore, even when sLOTUS and Aβ were added simultaneously, it was revealed that the number of living microglia increased by about 20% due to sLOTUS (Figure 4B). On the other hand, when treated with sLOTUS or Aβ for 4 hours in the same manner as the Phagocytosis Assay, it was confirmed that there was no change in the number of living cells compared to the control with the purified Tag of sLOTUS.

[0071] Also, the number of dead cells was measured by the LDH assay. Since LDH is released extracellularly when the cell membrane is damaged, it can be seen that the higher the absorbance, the more dead cells with a disintegrated cell membrane there are. As a result, it was revealed that the number of dead cells decreased by about 20% due to sLOTUS compared to the control with the purified Tag (Figure 4C). Furthermore, even when sLOTUS and Aβ were added simultaneously, it became clear that the number of dead microglia tended to decrease due to sLOTUS (Figure 4D). From the above results, it was revealed that sLOTUS improves the survival rate of microglia.

[0072] sLOTUS promotes the phagocytosis of Aβ by microglia Microglia have been reported to suppress the progression of the disease by removing Aβ in the brain through the Aβ clearance function (Ana Griciuc et al., Neuron. 2013 May 22;78(4):631-43. and Shweta Mandrekar et al., J Neurosci. 2009 Apr 1;29(13):4252-62.). Therefore, the relationship between LOTUS and phagocytic ability in microglia was examined. sLOTUS and Aβ-pHrodo were simultaneously added to primary microglial cells, and after 4 hours, the Aβ phagocytic ability of microglia was analyzed by the Phagocytosis Assay method. The Aβ-pHrodo used was taken up by microglia and emitted fluorescence in response to changes in the pH within lysosomes. Therefore, the number of Aβ phagocytosed was evaluated by quantifying the fluorescence. In fluorescence microscope images, more fluorescence was observed with sLOTUS compared to the control by the purified Tag of sLOTUS (image data is omitted). Therefore, as a result of quantifying the number of fluorescence, it was revealed that Aβ phagocytosis by microglia increased by approximately 20% with the addition of sLOTUS (Figure 5). Furthermore, the same tendency was confirmed even when phagocytosis was evaluated with Aβ-pHrodo after treating with sLOTUS similar to ELISA for 24 hours.

[0073] Example 3: Aβ antagonistic effect of LOTUS via PirB [Materials and Methods] Culture of COS-7 cells In African green monkey kidney-derived COS-7 cells, DMEM (4.5 g / l Glucose with L-glutamine and sodium pyruvate: Nacalai Tesqu) supplemented with 10% heat-inactivated FBS (Biological Industries) and 0.5% Penicillin / streptomycin (Nacalai Tesque) was used as the maintenance medium, and cultured in a 10 cm culture dish at 37°C under 5% CO2 conditions, and subcultured once every 3 days.

[0074] Gene transfer into COS-7 cells by Lipofection method COS-7 cells were seeded in a 4-well culture plate (Thermo Scientific) at 5.0×10 4 cells / well with a total medium volume of 400 μl and incubated until the cells adhered (37 °C, 5% CO2, 4 hours). After confirming cell adhesion, plasmids were introduced using FuGENE6 Transfection Reagent (Roche) to forcibly express mouse PirB (NM_001357394.1, SEQ ID NOs: 16, 17), mouse LOTUS (NM_145123.5, SEQ ID NOs: 18, 19), human LilrB2 (NM_001080978.4, SEQ ID NOs: 20, 21), and human LOTUS (NM_001206528.3, SEQ ID NOs: 1, 2). The expression of each protein was confirmed by immunostaining.

[0075] Immunostaining Immunostaining was performed to confirm the expression of each protein. Primary antibody reactions (37 °C, 5% CO2, 1 hour) were carried out using anti-LOTUS (R&D) and anti-PirB IgG (R&D), and anti-LilrB2 (R&D) diluted 200-fold in maintenance medium 44 hours after plasmid addition. After the reaction, the samples were fixed with 4% PFA / PBS (room temperature, 1 hour, protected from light), and secondary antibody reactions (room temperature, protected from light) were carried out using Alexa Fluor 488 anti-Goat IgG (Jackson ImmunoResearch) and Alexa Fluor 596 anti-goat IgG (Jackson ImmunoResearch). The stained samples were observed and imaged for fluorescence using a BZ-8100 microscope (Keyence).

[0076] Experiment on inhibition of Aβ-PirB binding by LOTUS COS-7 cells were co-expressed with mouse PirB and mouse LOTUS, or human LilrB2 and human LOTUS by the above Lipofection method. After 44 hours of plasmid addition, polymerized Aβ was diluted with maintenance medium to each concentration (100 nM, 200 nM, 500 nM) and added, and then incubated (37 °C, 5% CO2, 1 hour). After fixation with 4% PFA / PBS (room temperature, 1 hour, light-shielded), activation treatment (67 °C, 1 hour) was performed. Furthermore, AP (Alkaline Phosphatase) was added to Aβ by the ABC method (Avidin-Biotinylated enzyme Complex method). A substrate mixture of NBT / BCIP (4-Nitoro blue tetrazolium chrolide / 5-Bromo-4-chloro-3-indolyl-phosphatase: Roche) for the added AP was added to perform a color reaction. Cells to which Aβ was bound were colored dark blue. The samples after color development were observed and imaged in bright field by a BZ-8100 microscope (Keyence). Furthermore, for quantification of binding, a pNPP solution (1 mg / ml p-Nitrophenyl phosphate) was added as a substrate for the AP added to Aβ by the above method, and the absorbance at 405 nm was measured using a microplate reader X MarkTM (BioRad) 4 hours later. The expression of each protein was confirmed by immunostaining.

[0077] animal C57BL / 6J wild-type mice (WT) were purchased from Japan SLC. The transgenic mice overexpressing the lotus gene (LOTUS-tg) were generated by microinjecting into the pronuclear stage fertilized eggs of C57BL / 6J mice a sequence consisting of the Synapsin-1 promoter specifically expressed in the axons of nerve cells, the Igk chain secretion signal, the HA tag gene, the mouse lotus gene (the region encoding aa29 - 646 of mouse LOTUS, positions 240 - 2096 of SEQ ID NO: 18), and the Rabbit β-globin (Ex2 - Ex3) intron / polyA sequence. All mice were provided with a standard mouse breeding environment, autoclaved feed, and water.

[0078] Primary culture method of hippocampal neurons The hippocampus was dissected from mouse embryos (E17.5) and dispersed at 37°C for 12 minutes using 0.25% Trypsine and 100 μg / ml DNase. The dispersed cells were seeded at 5×10 4 cells / well in a 24-well plate with a glass coverslip (φ12 mm: Matsunami) placed on the bottom. The glass coverslip was surface-treated with a 100 μg / ml Poly-L-Lysin solution before seeding. Neurobasal medium (gibco) and 10% FBS (biowest) were used as the plating medium, and 1xB-27 (gibco), 1xGlutamax (gibco), and Neurobasal medium (gibco) were used as the maintenance medium.

[0079] Gene transfection into primary cultured hippocampal neurons by Lipofection An experiment was conducted to forcibly express EGFP by transfection in primary cultured cells in order to clearly observe the cell morphology. Gene transfection was performed on primary cultured cells on the 11th day of culture (DIV11). In a 1.5 ml tube, 1.5 μl of Viafect Transfection reagent (Promega) and 0.5 μg of plasmid DNA were mixed with 50 μl of Neurobasal medium and allowed to stand (room temperature, 15 minutes). Then it was added to a 24-well plate seeded with cells and incubated (37 °C, 5% CO2, 3 hours). After incubation, the medium was removed and the remaining reagents were washed with HBSS warmed to 37 °C, and 400 μl / well of maintenance medium was added and incubated until each treatment (37 °C, 5% CO2).

[0080] Quantification of spine density Primary cultured cells 1 day after gene transfection (DIV12) were treated with Aβ at each concentration (0 nM, 200 nM, 500 nM) (37 °C, 5% CO2, 2 days). The treated cells were fixed and mounted in the same manner as above. Imaging was also performed in the same manner as above. Spines within a range of 50 μm from the branch of the dendrite closest to the cell body were measured. Spines that protruded more than 0.5 μm from the dendrite and had a width of the protruding part of 0.5 μm or more were counted as spines. The number of spines per 10 μm of the analyzed dendrite was defined as the spine density.

[0081] [Results] LOTUS suppresses the binding of Aβ and PirB To examine the effect of LOTUS on the binding of Aβ to PirB, experiments were conducted using COS-7 cells co-expressing PirB and LOTUS. First, the binding of Aβ to PirB was confirmed as previously reported (Kim T, et al. Science. 2013;341(6152):1399-404.). When cells overexpressing PirB were treated with Aβ at various concentrations (100 nM, 200 nM, 500 nM), the binding (color development) intensity of Aβ increased in a concentration-dependent manner (Figure 6a). In cells co-expressing LOTUS and PirB, the binding of Aβ was decreased compared to that in cells expressing PirB alone. Also, to confirm whether LOTUS affects the expression level of PirB, the expression of PirB in each group was confirmed by the pNPP method. As a result of the experiment, no difference was observed in the expression level of PirB between the single expression of PirB and the co-expression of LOTUS+PirB (Figure 6b). From this, it was found that in this experiment, LOTUS inhibits the binding of Aβ-PirB without affecting the expression level of PirB. Next, experiments were conducted using pNPP for quantitative analysis. pNPP turns yellow when reacting with AP. Since the AP activity can be quantified by measuring the absorbance at 405 nm of the colored solution, the binding of Aβ can be quantified. In the pNPP method, the binding of Aβ was suppressed by approximately 20% in the co-expression of LOTUS and PirB compared to the single expression of PirB (Figure 6c). From these results, it was found that LOTUS inhibits the binding of Aβ to PirB.

[0082] LOTUS inhibits the decrease in spine density caused by Aβ The spine is a structure on the postsynaptic side of the synapse, which is the junction between nerve cells. In Alzheimer's disease (AD), it is thought that the decrease in spine density of nerve cells in the brain leads to a decline in neural plasticity and cognitive function (Querfurth HW, LaFerla FM. N Engl J Med. 2010, 362:329-44.; and Mucke L, Masliah E, Yu GQ. J Neurosci. 2000, 20:4050-8.). In primary cultured hippocampal neurons of WT mice, the spine density decreased in a concentration-dependent manner upon the addition of Aβ (Figures 7a, b). In contrast, in primary cultured neurons of LOTUS-tg mice, the decrease in spine density due to Aβ was suppressed (Figures 7a, b). From these results, it was found that LOTUS suppresses the decrease in spine density caused by Aβ.

[0083] Human LOTUS inhibits the binding of Aβ to LilrB2 (human PirB) For the purpose of analyzing the function of LOTUS in humans, a binding experiment of Aβ was conducted in the same manner as in Figure 6 to verify whether human LOTUS has an antagonistic effect on LilrB2 (NM_001080978.4, SEQ ID NO: 20, 21), which is the human homolog of PirB. LilrB2 was expressed in COS-7 cells, and Aβ was treated at each concentration (100 nM, 200 nM, 500 nM). As a result, the binding (color development) intensity of Aβ increased in a concentration-dependent manner (Figure 8a). In cells co-expressing human LOTUS and LilrB2, the binding of Aβ was significantly decreased compared to that in cells expressing LilrB2 alone. In addition, to confirm whether human LOTUS affects the expression level of LilrB2, the expression level of LilrB2 in each group was examined, and no difference in the expression level was found between the single expression of LilrB2 and the co-expression of human LOTUS and LilrB2 (Figure 8b). From this, it was found that in this experiment, human LOTUS inhibits the binding of Aβ-LilrB2 without affecting the expression level of LilrB2. Next, an experiment was conducted using pNPP for quantitative analysis. According to the pNPP method, the binding of Aβ was inhibited by approximately 40% in the co-expression of LOTUS and LilrB2 compared to the single expression of LilrB2 (Figure 8c). From these results, it was found that human LOTUS inhibits the binding of Aβ and LilrB2.

[0084] Example 4: Promoting effect on synapse formation in LOTUS overexpressing (LOTUS-Tg) mice [Materials and methods] Primary culture method of hippocampus The primary culture method of hippocampal neurons was partially modified with reference to existing reports (Banker G.A. and Cowan W.M. Brain Res. 126(3), 397-425, 1977.). Mice of each genotype of WT and LOTUS-TG were deeply anesthetized with isoflurane (Pfizer), and the fetuses (E17.5) were removed. The hippocampus was removed and dispersed at 37 °C for 12 minutes using 0.25% trypsin and 100 μg / ml DNase. The dispersed cells were 0.5×10 5Cells were seeded in a 24-well dish at a density of cells / well. Neurobasal medium (Gibco) containing 10% FBS (Biowest) was used as the cell culture introduction medium, and 1×B-27 (Gibco), 1×Glutamax (Gibco), and Neurobasal medium (Gibco) were used as the maintenance medium, respectively.

[0085] Fluorescent immunocytochemistry On the 14th day of culture, primary hippocampal cultured cells were fixed with methanol at -20°C for 8 minutes and blocked with 1% BSA / PBS for 20 minutes. Then, Mouse anti-PSD-95 antibody (1 / 1000), Rabbit anti-Bassoon antibody (1 / 1000), and Chicken anti-MAP2 antibody (1 / 1000) diluted with 1% Bovine Serum Albumin (BSA: Nacalai Tesque) / PBS were used as primary antibodies. After reacting the primary antibodies at room temperature for 1 hour, the cells were washed with PBS, and Alexa Fluor488 Goat anti-Mouse IgG (1 / 2000), Alexa Fluor 594 Goat anti-Rabbit IgG (1 / 2000), and Alexa Fluor 647Goat anti-Chicken IgY (1 / 2000) diluted with 1% BSA / PBS were used as secondary antibodies, and the secondary antibody reaction was performed at room temperature for 1 hour. Then, the cells were washed with PBS and mounted using Fluoromount (Cosmo Bio).

[0086] Analysis of synaptic density in primary hippocampal cultured cells All fluorescent immunostaining images were acquired using a confocal microscope (TCS SP8: Leica) and Las X software (Leica). Granules with co-localization of PSD-95 and Bassoon within 40 μm from the branching point closest to the cell body of MAP2-positive dendrites were counted, and the synaptic density per 10 μm of dendrites in each cell was measured.

[0087] Analysis of dendritic spine density using mouse brain sections Male mice (2 months old) were deeply anesthetized with isoflurane and perfused with 4% PFA / PBS. Then, the brains were removed and immersed in 4% PFA / PBS overnight. After that, the brains were immersed in 30% sucrose. Subsequently, 30-μm-thick coronal sections were prepared using a cryostat. Fluorescent images were acquired using a confocal microscope (TCS SP8) with LAS X software. Spine density was measured semi-automatically using Neuron Studio software (Rodriguez, A., et al., PLOS ONE 3 e1997, (2008)). Spine density was measured with thin type having a head-to-neck diameter ratio of 1.1 or more and a maximum head diameter of 0.35 μm or less, mushroom type having a head-to-neck diameter ratio of 1.1 or more and a maximum head diameter of 0.35 μm or more, and stubby type having a head-to-neck diameter ratio of 1.1 or less. The spine density at the first branching site of the apical dendrite or basal dendrite from hippocampal CA1 pyramidal neurons was quantified.

[0088] Behavioral analysis ·Social cognitive memory task The social cognitive memory task is a behavioral analysis that evaluates social cognitive memory, which is hippocampus-dependent memory (Kogan, J. H., et al., Hippocampus 10, 47-56, (2000); Ishikawa, R., et al., Hippocampus 24, 784-793, (2014)). First, as training, naïve mice (male, 2-3 weeks old) that had never met before were allowed to meet with adult mice (male, 2 months old) whose memory ability was to be evaluated for 3 minutes in the cage of the adult mice, and the time (I.T.: Investigation time) during which the adult mice touched the nose of the naïve mice and sniffed was measured as the time required for individual recognition. After 24 hours, as a test, I.T. was measured again using the same combination of adult and naïve mice. If the I.T. at the test was significantly decreased compared to that at the training, it was judged that the adult mice remembered the naïve mice.

[0089] · Morris water maze task The Morris water maze task was conducted to evaluate spatial-dependent memory, which is hippocampus-dependent memory (Morris, R. G. Learn. Motiv. 12, 239-260, (1981).). Male mice (2 months old) were trained twice a day for 1 minute for a total of 6 days to learn spatial memory. Tests were conducted 24 hours after the training on the 3rd and 6th days. During the test, the platform was removed, and the mice were allowed to swim freely for 1 minute. The residence time in each section during the test was measured to evaluate the memory formation ability.

[0090] Statistical analysis Verification of statistical significance was performed using J-STAT software. Two-way repeated ANOVA was used for the escape latency of the Morris water maze task. Also, the residence time in the TQ section of the Morris water maze task was analyzed using 2 a χ test and one-way ANOVA with post-hoc Steel-Dwass.

[0091] [Results] LOTUS antagonizes Nogo and increases the spine density of cultured hippocampal neurons Previous reports have shown that Nogo decreases synaptic density in primary cultured hippocampal neurons (An et al., Journal of Cell Science, 2016, 129(6), 1198 - 1209). LOTUS is expressed in the synaptic region including both the presynaptic and postsynaptic parts, and it has been reported that the synaptic density decreases when LOTUS is deficient (Nishida et al., Scientific reports, 2021, 11(1), 1 - 9). Therefore, by immunostaining with the postsynaptic protein marker PSD - 95, we investigated whether exogenously introduced LOTUS antagonizes Nogo and affects spine density. Although there is also a membrane - bound form of LOTUS, in this experiment, the soluble form of LOTUS (s - LOTUS) was used for exogenous introduction. Cultured hippocampal neurons from WT mice at DIV12 were treated with Nogo - 66 and s - LOTUS, and after 48 hours, double - immunostained with the PSD - 95 - positive sites on dendrites and MAP2, which is a dendrite marker, and measured as synaptic sites (microscopic image data is omitted). Administration of Nogo - 66 decreased the spine density of PSD - 95 compared to the control (Figure 9). Co - administration of Nogo - 66 and s - LOTUS restored the decrease in PSD - 95 - positive spine density to the same level as the control. Interestingly, the PSD - 95 - positive spine density also increased with s - LOTUS alone. These results indicate that s - LOTUS antagonizes Nogo and thereby increases the spine density of cultured hippocampal neurons.

[0092] Overexpression of LOTUS increases the synaptic density of cultured hippocampal neurons LOTUS binds to NgR1 and suppresses Nogo signaling (Sato et al., 2011, Science, 333(6043), 769-773.). A decrease in Nogo signaling is known to increase synaptic density (Wills et al., Neuron, 2012, 73(3), 466-481.; Zagrebelsky et al., Neurobiology of learning and memory, 2017, 138, 154-163.). Therefore, the effect of LOTUS overexpression on synaptic density was examined. Primary cultured hippocampal neurons from WT mice and LOTUS-Tg mice were immunostained with PSD-95 as a postsynaptic marker, bassoon as a presynaptic marker, and MAP2 as a dendritic marker at DIV14, and synapses on dendrites were identified to measure synaptic density (Figures 10a-d). As a result, the synaptic density was significantly increased in neurons of LOTUS-Tg mice compared with WT mice (Figure 10e). These results indicate that LOTUS overexpression promotes synaptogenesis and increases synaptic density.

[0093] The spine density is increased in the hippocampal CA1 region of LOTUS-Tg mice Since the deficiency of LOTUS reduces spine density in the hippocampal CA1 region (Nishida et al., supra 2021), overexpression of LOTUS may also affect spine density. Therefore, we investigated the effect of LOTUS overexpression on dendritic spine density and its morphology in the hippocampal CA1 region of adult mice at 2 months of age. WT mice and LOTUS-Tg mice were crossed with Thy1-EGFP mice, and dendritic spines of WT mice and LOTUS-Tg mice were examined by observing EGFP-positive dendrites. The total number of spines in the dendrites on the apical side of hippocampal CA1 pyramidal neurons was higher in LOTUS-Tg mice than in WT mice (Figures 11a, b). Furthermore, in LOTUS-Tg mice, the number of thin-type and mushroom-type spines was also higher than in WT mice, but there was no difference in the number of stubby-type spines (Figures 11a, b). The total number of spines was also increased in LOTUS-Tg mice. Similar measurements were also performed on neurons on the basal side of dendrites, and the results were the same (Figures 11c, d). These results indicate that overexpression of LOTUS increases spine density in the hippocampal CA1 region of young adult mice.

[0094] Overexpression of LOTUS enhances memory To investigate whether overexpression of LOTUS affects learning and memory functions, we first conducted a social recognition test. This behavioral analysis evaluates hippocampus-dependent social recognition memory formation in mice. In this experiment, mature test mice (2 months old) were allowed to interact with unfamiliar young mice for 1.5 minutes, and 24 hours later, it was determined whether the test mice remembered the young mice. As a result, the investigation time on the second day was significantly reduced in LOTUS-Tg mice compared to the first day, whereas no significant decrease was observed in WT mice (Figure 12a). Furthermore, the recognition index, which is the ratio of the social investigation time on the second day to the social investigation time on the first day, was lower in LOTUS-Tg mice than in WT mice, suggesting that memory formation was enhanced in LOTUS-Tg mice compared to WT mice (Figure 12b). These results suggest that overexpression of LOTUS enhances social recognition-related memory in mice.

[0095] Next, the Morris water maze test was used to evaluate hippocampus-dependent spatial learning and memory. In LOTUS-Tg mice, after 3 days of training, with the platform removed, the residence time in the TQ area was significantly longer than that in other areas (chi-square test; Figure 13a). On the other hand, no difference was observed in WT mice. Furthermore, the proportion of time spent in TQ was significantly higher in LOTUS-Tg mice than in WT mice (Figure 13b). There was no difference in the body weight and swimming speed between LOTUS-Tg mice and WT mice. These results suggest that spatial learning and memory are enhanced in LOTUS-Tg mice.

[0096] From the above results, it was suggested that overexpression of LOTUS may enhance hippocampus-dependent memory formation abilities such as social cognitive memory and spatial learning.

[0097] Overexpression of LOTUS prevents memory dysfunction associated with aging It has been reported that the expression level of LOTUS decreases with aging, and memory impairment develops accordingly (VanGuilder et al., 2013). Furthermore, we have clarified that LOTUS-Tg mice enhance memory (Figures 12 and 13). Therefore, we investigated whether LOTUS-Tg mice have less memory impairment associated with aging. The expression of LOTUS in the hippocampus of aged mice was examined by Western blotting. As a result, it was found that the expression level of LOTUS in WT mice was significantly lower in 18-month-old (aged) mice than in 2-month-old (young) mice (Figures 14-1a, b). However, no significant difference was observed in the expression level of LOTUS in LOTUS-Tg mice (Figures 14-1a, c). The LOTUS expression level in aged LOTUS-Tg mice was approximately 1.5 times that of young WT mice (Figure 14-1c). On the other hand, when the expression level of NgR1 in the hippocampus was quantified, the expression level of NgR1 tended to decrease with aging in both WT mice and LOTUS-Tg mice. Furthermore, no age-related change was observed in the expression level of Nogo in WT mice and LOTUS-Tg mice (Figure 15).

[0098] To evaluate the relationship between the decrease in LOTUS expression level and the change in spine density in the hippocampal CA1 region with aging, the spine densities of WT mice and LOTUS-Tg mice were compared. As a result, in old-aged LOTUS-Tg mice, higher spine densities were shown in both apical and basal dendritic protrusions compared to old-aged WT mice (Figure 14-2d-g).

[0099] Next, the hippocampus-dependent social memory of old-aged mice was evaluated by a social cognition test. In this experiment, young WT mice at 2 months of age can acquire memory in a 3-minute experiment (Nishida et al., 2021). As a result, in old-aged WT mice at 1.5 years of age, there was no difference in the investigation time between day 1 and day 2, suggesting cognitive memory impairment in old-aged WT mice (Figure 14-3h). However, in LOTUS-Tg mice, a significant decrease in the investigation time was observed. Since this was observed not only in young mice but also in old-aged mice, it was suggested that old-aged LOTUS-Tg mice do not have hippocampus-dependent cognitive memory impairment (Figure 14-3i).

[0100] From these results, it was suggested that old-aged LOTUS-Tg mice retain the ability of hippocampus-dependent memory formation because the expression of LOTUS and the spine density in the hippocampal CA1 region are maintained. Thus, it is inferred that overexpression of LOTUS helps maintain the LOTUS level in the aging brain, thereby preventing a decrease in spine density in the hippocampus and senile memory impairment (senile amnesia) associated with aging.

[0101] Example 5: Novel Object Recognition Test (NORT) [Materials and Methods] APP NL-G-F mice Knock-in mice with gene mutations inserted into the amyloid-β region of the mouse App gene. In addition to humanizing the mouse amyloid-β sequence, single Swedish mutations, double mutations with Iberian mutations, and triple mutations with Arctic mutations of familial Alzheimer's disease mutations were introduced. Homozygous mice are viable and fertile. The production ratio of toxic amyloid-β species (Aβ42) is increased, and amyloid plaques are formed from an early age. APP NL-G-F homozygous mice form amyloid plaques in the brain from 2 months of age. Along with the formation of amyloid plaques, neuroinflammation and synaptic loss are observed. Furthermore, APP NL-G-F knock-in mice show abnormal behavior in the Y-maze from 6 months of age. An important model mouse in Alzheimer's disease (AD) research.

[0102] Novel object recognition test (schematic diagram shown in Figure 16) Mice at 12 months of age (wild-type mice, APP-KI mice, and APP / LOTUS-Tg mice) were used for the test. Day1 Each mouse was placed separately in a cage and left standing for 1 hour. The same object was placed in the field (460 mm × 460 mm), and the mouse was allowed to freely explore it for 10 minutes and then returned to the breeding cage. After each individual analysis, the field and the object were cleaned with 70% ethanol. Day2 Each mouse was placed separately in a cage and left standing for 1 hour. The same object as on Day1 was placed in one corner of the field, and a new object was placed in the other corner. The mouse was allowed to freely explore them for 10 minutes and then returned to the breeding cage. After each individual analysis, the field and the object were cleaned with 70% ethanol. The exploration time for the object was measured to evaluate the memory function.

[0103] [Results] In wild-type (WT), the exploration time for the novel object was significantly longer than that for the familiar object on Day1 on Day2, confirming that the object was remembered.

[0104] In AD mice (APP-KI mice), there was no difference in the exploration time for two objects, indicating that they did not remember the objects. On the other hand, in mice obtained by crossing AD mice and LOTUS-overexpressing mice (APP / LOTUS-Tg), it was found that they remembered the objects in the same way as WT mice.

[0105] From these results, it was found that increasing the expression level of LOTUS improved the decline in memory function confirmed in APP-KI mice. That is, LOTUS was found to improve the AD-like pathological conditions (memory impairment) observed in AD mice.

[0106] Production Example 1: Production of Excitatory Neurons Derived from Human Pluripotent Stem Cells <Cell> As human pluripotent stem cells (hPSC), commercially available products or those prepared by oneself are used. <Kit to be Used> Quick-Neuron (trade name) Excitatory - SeV Kit (Small) (ELIXIRGEN SCIENTIFIC) Components of the kit: QN-SeV, Component N, Component P, Component G1, Component G2, and Coating Agent A <Required Reagents> Prepare the reagents described in Table 3. The cell dissociation reagent Solution D1 is purchased from ELIXIRGEN SCIENTIFIC.

[0107]

Table 3

[0108] <Medium to be Prepared in Advance> Medium N (composition as shown in Table 4)

[0109]

Table 4

[0110] <Method> I. Coating of plates (performed 3 days before culturing) 1. Mix the following components in a 15 ml conical tube to prepare diluted coating agent A. (6.0 μl of coating agent A, 1.8 ml of PBS at 4°C) 2. Add 400 μl / well of the diluted coating agent A to each well. 3. Incubate the plate at 4°C (4°C, 3 Days).

[0111] II. Day0 1. Mix the following components in a 15 ml conical tube to prepare Medium iN (2.5 ml of Medium N, 2.5 μl of ROCK inhibitor). Store the remaining Medium N at 4°C for later use. 2. Thaw QN-SeV-P on ice. 3. Drain the old medium from the hPSC culture, and add 2 ml of PBS to each well to be collected. 4. Shake the plate 3 times, aspirate the PBS from the culture solution, and add 300 μl / well of cell dissociation reagent Solution D1 to each well. Store the remaining Solution D1 at 4°C for use on day 3. 5. Incubate the culture plate at 37°C, 5% CO2 for 5 minutes. 6. Remove Solution D1, and add 1 ml of Medium iN to the well. 7. Pipette the medium on the bottom of the well 8 - 15 times to disperse and detach the cells. 8. Using the same pipette tip, collect the cell suspension into a 1.5 ml tube. 9. Count the cells and measure the viability. 10. Take out the required amount (total 2.2x10 5 cells) of cell suspension for 4 wells, and add 10% extra. Transfer this cell suspension to a 15 ml conical tube. 11. Adjust the volume of the cell suspension to 110 μl with Medium iN. 12. Add 55 μl of QN-SeV-P to hPSCs and tap 2-3 times with your finger to mix. 13. Incubate the cell suspension at 33 °C and 5% CO2 for 10 minutes. Tap with your finger every minute. 14. Make the cell suspension up to 1.1 ml with Medium iN. 15. Aspirate the diluted coating agent A one well at a time and add 250 μl of the cell suspension. 16. Quickly move the plate back and forth and from side to side 5 times each to evenly disperse the treated cells. Disperse the treated cells evenly in the culture medium. 17. Incubate the culture plate overnight at 33 °C and 5% CO2.

[0112] III. Day1 1. Prepare Medium N (G1) by mixing the following components in a 15 ml conical tube. (Medium N 4.4 ml, Component G1 of the kit 8.8 μl) 2. Add puromycin to Medium N (G1) at an appropriate concentration. 3. Pipette out most of the medium from each well and add 500 μl of Medium N (G1) with puromycin added. 4. Incubate the culture plate overnight at 33 °C and 5% CO2.

[0113] IV. Day2 1. Warm Medium N (G1) with puromycin added at room temperature for 20 - 30 minutes. 2. Remove most of the medium from each well and add 500 μl of Medium N (G1) with puromycin added. 3. Incubate the culture plate overnight at 37 °C and 5% CO2.

[0114] V. Day3 1. Immerse the tips of a 12 mm glass coverslip and forceps in 100% ethanol for 3 minutes. 2. Place one cover slip at a time, air dry for 1 minute or until completely dry, and place it in the wells of a 24-well plate using sterilized ethanol. 3. Vortex ornithine briefly and centrifuge at maximum speed for 1 minute. 4. Add 300 μl of ornithine to the surface of each glass cover slip or well. 5. Incubate the plate at 37 °C and 5% CO2 for at least 2 hours. 6. Thaw laminin and add 15 μl of laminin to cold PBS. Mix well. Note: All PBS washes are performed by dropping room temperature PBS. Cold PBS is only used for coating. 8. Aspirate the supernatant from each cover slip / well and add 500 μl of PBS. 9. Repeat step 8. 10. Aspirate PBS from each cover slip / well and add 300 μl of diluted laminin. 11. Incubate the plate at 37 °C and 5% CO2 for at least 2 hours. 12. While laminin is incubating, prepare Medium N (G2P) (5.5 ml of Medium N, 5.5 μl of Component G2 of the kit, and 2.8 μl of Component P). Place it in a 15 ml conical tube. 13. After incubation of laminin, remove from each cover slip or well 14. Wash with 500 μl of PBS × 2 15. Add 300 μl of Medium N (G2P) to each well or cover slip. 16. Incubate the plate at 37 °C and 5% CO2 until the cells are ready for plating. 17. Remove the old medium from the well by pipetting and add 500 μl of PBS. 18. Withdraw PBS from the well and add 80 μl of Solution D1. 19. Shake the plate 3 times to spread D1 evenly. 20. Incubate the culture plate at 37 °C and 5% CO2 for 3 minutes. 21. Carefully aspirate Solution D1 from the well using a P200 pipettor and add 200 μl of Medium N (G2P). 22. Pipette 6 - 8 times using a P200 pipettor to detach the cells and quickly disperse the medium on the bottom of the well. 23. Using a P200 pipettor, slowly pipette the cell suspension up and down up to 5 times within the well to break up cell aggregates. 24. Using a P200 pipettor set to 100 μl, collect 200 μl of the cell suspension from the well and transfer 100 μl each to a glass coverslip coated with ornithine and laminin. 25. Incubate the culture plate overnight at 37 °C and 5% CO2.

[0115] VI. Day4 1. Aspirate the medium from each well and add 800 μl of Medium N (G2P). 2. Incubate at 37 °C and 5% CO2 for 3 days.

[0116] VII. Day7 1. Aspirate 400 μl of the old medium from each well and add 400 μl of Medium N (G2P) each. 2. Incubate at 37 °C and 5% CO2 for 3 days.

[0117] VIII. Day10 It can be used as mature excitatory neurons. After day 10, the differentiated neurons can be maintained in a maintenance medium (recommended: Quick - Neuron (trade name) Excitatory - Maintenance Medium from ELIXIRGEN SCIENTIFIC, catalog number EX - MM). Differentiation into excitatory neurons can be confirmed using anti - TUBB3 antibody and anti - vGLUT1 antibody.

[0118] Production Example 2: Production of Human Pluripotent Stem Cell - Derived Oligodendrocyte Precursor Cells Oligodendrocyte progenitor cells (OPCs) derived from human pluripotent stem cells (hPSCs) can be produced by the method described in Douvaras and Fossati, Generation and isolation of oligodendrocyte progenitor cells from human pluripotent stem cells. Nat Protoc 10, 1143-1154 (2015). The specific production method is described below.

[0119] <Preparation of medium> mTeSR1 medium Penicillin-streptomycin and mTeSR1 supplement (StemCell Technologies, cat. no. 05850) are added to the mTeSR basal medium to make the final concentration of all components 1x.

[0120] mTeSR custom medium mTeSR custom supplement (StemCell Technologies, customized) and penicillin-streptomycin are added to the mTeSR basal medium to make the final concentration of all components 1x.

[0121] Neural induction medium 10 μM SB431542, 250 nM LDN193189 and 100 nM RA are added to the mTeSR custom medium on the day of use. The mTeSR custom medium can be replaced with a basal medium containing 25 μg / ml insulin.

[0122] Basal medium NEAA (MEM non-essential amino acids solution, 100×; Life Technologies, cat. no. 11140-050), GlutaMAX (100×; Life Technologies, cat. no. 35050079), 2-mercaptoethanol and penicillin-streptomycin were added to DMEM / F12 (Life Technologies, cat. no. 11320082) to make the final concentration of each component 1x.

[0123] N2B27 medium N2 supplement (Life Technologies, cat. no. 17502-048) and B27 supplement (Life Technologies, cat. no. 12587-010) were added to basal medium to make the final concentration of each component 1x. 25 μg / ml insulin was added. 100 nM RA and 1 μM SAG were added on the day of use.

[0124] Glial medium It is prepared with the following composition.

[0125]

Table 5

[0126] <Differentiation induction method> Procedure 1: Preparation of hPSCs (4 - 5 days) [1] Partially thaw a cryovial (1 mL volume) containing hPSCs in a 37°C water bath over 1 - 2 minutes. [2] Slowly add 1 mL of mTeSR1 medium into the cryovial and transfer the total 2 mL cell suspension to a 15 mL tube. [3] Add mTeSR1 medium to the above 15 mL tube and make up to 10 mL. ↓ Centrifuge at 200g for 4 minutes, 20 - 23°C (room temperature) [4] Remove the supernatant and resuspend the cell pellet in 2 mL of mTeSR1 + 10 μM Y27632 (ROCK inhibitor) medium. [5] Seed the suspended cells onto a 6-well plate coated with Matrigel (BD Biosciences, cat. no. 354277). ↓ Incubate at 37 °C in a 5% CO2 incubator for 24 hours [6] Remove the medium and add 2 mL of mTeSR1 medium. [7] Incubate in the same way and change the medium once a day. [8] When 70 - 90% confluent, remove the medium and add 1 mL of pre-warmed Accutase (Life Technologies, cat. no. A11105-01). ↓ Incubate at 37 °C in 5% CO2 for 5 minutes [9] Add 2 mL of DMEM / F12 medium (Life Technologies, cat. no. 11320082).

[10] Gently pipette 2 - 5 times with a p1000 pipetteman to fully dissociate the hPSC colonies into single cells.

[11] Add the cell suspension to a 15 mL tube and further add 5 mL of DMEM / F12. ↓ Centrifuge at 200 g for 4 minutes at 20 - 23 °C (room temperature)

[12] Remove the supernatant and resuspend the cell pellet in 1 mL of mTeSR1 + 10 μM Y27632 medium. ↓ Count the number of cells

[13] Prepare a Matrigel-coated 6-well plate and add 1.5 mL of mTeSR1 + 10 μM Y27632 medium.

[14] Seed 8×10 4 ~1×10 5 cells per well. ↓ Incubate at 37 °C in a 5% CO2 incubator for 24 hours

[15] Remove the medium and add 2 mL of mTeSR1 medium.

[16] Culture the cells until the diameter of the hPSC colonies reaches 100 - 250 μm (1 - 2 days).

[0127] Procedure 2: OLIG2 + Inducing differentiation into progenitor cells (12 days)

[17] Once the hPSC colonies reach a diameter of 100 - 250 μm, remove the medium and add 2 mL of Neural induction medium to each well. (This is day 0 at this point) * By adding RA at a low concentration (100 nM) from day 0 of differentiation induction, the yield of OLIG2 + progenitor cells is significantly improved. ↓ Culture in a 37°C 5% CO2 incubator for 8 days * Change the medium once a day. At this time, use fresh RA, SB431542, and LDN193189 each time.

[18] Exchange the medium with N2 medium. Change the medium once a day. * By day 8, the cells become confluent, and by day 12, the cells form a 3D structure. The 3D structure can be clearly confirmed and becomes an important checkpoint for subsequent differentiation operations. * When changing the medium, use fresh RA and SAG each time. * Due to the rapid expansion of the cells and the increase in cell number, the medium becomes yellowish. (Medium change is necessary every day to appropriately supply the nutrients required by the cells!)

[0128] Procedure 3: Cell separation and formation of OLIG2 - enriched aggregates - OLIG2 after enzymatic digestion into single cells + Cell aggregation (18 days) → Only OLIG2+ cells aggregate into spheres

[19] Remove the medium and add 1 mL of pre - warmed Accutase. ↓ Incubate at 37°C in 5% CO2 for 5 minutes

[20] Add 2 mL of DMEM / F12 medium.

[21] Gently pipette 2 - 5 times with a p1000 pipetteman to make single cells sufficiently.

[22] Add the cell suspension to a 15 mL tube, and then add another 5 mL of DMEM / F12. ↓ Centrifuge at 200g for 4 minutes at 20 - 23°C (room temperature)

[23] Remove the supernatant, and add 1 mL of N2B27 medium to resuspend the cell pellet.

[24] Dispense the cell suspension into 2 wells of an ultra - low attachment 6 - well plate.

[25] Add 3 mL of N2B27 medium. ↓ Culture in an incubator at 37°C in 5% CO2 for 2 days

[26] Add the entire amount of medium containing cell aggregates into a 15 mL tube. ↓ Centrifuge at 100g for 2 minutes at 20 - 23°C (room temperature)

[27] Remove 2 / 3 of the medium, and add 2 mL of N2B27 medium.

[28] Return the aggregates to the same ultra - low attachment plate, and redistribute the number of aggregates using a pipette so that each well has approximately the same number.

[29] Perform operations

[26] -

[28] every other day until DIV - 20 when differentiation is reached.

[30] On the 20th day, add the cell aggregates to a 15 mL tube and wait for about 3 minutes (until the aggregates sink to the bottom of the tube).

[31] Remove 2 / 3 of the medium, and add PDGF medium.

[32] Gently pipette 5 times with a P1000 pipetteman.

[33] Return the aggregates to the same ultra - low attachment plate, and redistribute the number of aggregates using a p1000 pipette so that each well has approximately the same number.

[34] Perform operations

[30] -

[33] every other day until the 30th day.

[0129] Procedure 4: Selection and plating of aggregates (1 - 4 hours)

[35] On the 30th day, under aseptic conditions, use a microscope and a p200 pipetteman to collect round aggregates with a diameter of 300 - 800 μm that appear black at the center and golden or brown in color.

[0130] Procedure 5: Differentiation into OPCs by adherent culture (15 - 60 days)

[36] Plate at 20 spheres / well in a 6 - well plate coated with Poly - L - ornithine and Laminin. → 1 cm 2 Plate 2 spheres per cm (no passage required, confluent across the well around the 60th day).

[37] Add 3 mL of glial medium and rotate the plate to disperse the aggregates. ↓ Incubate in a 37°C 5% CO2 incubator

[38] Every other day, carefully replenish 2 / 3 of the medium with fresh glial medium until the 55th day. → By the 55th day, O4 + Cells can be visualized by live O4 staining or isolated by FACS for O4 cells. → The culture is kept in glial medium until the 75th day to enhance the efficiency of O4+ cells. → Perform medium changes as gently as possible. Do not tilt the plate to aspirate the old medium so that the cell aggregates are always covered with liquid. When aspirating the old medium, place the P1000 pipetteman near the wall of the well and aspirate gently without detaching the cells. Especially after the 40th day, avoid vibrating or moving the plate as much as possible to prevent cell detachment.

Claims

1. A prophylactic or therapeutic agent for Alzheimer's disease, comprising as an active ingredient a Crtac1B protein, a vector capable of expressing the Crtac1B protein, or a cell expressing the Crtac1B protein.

2. An agent for enhancing memory and / or cognitive function in a patient in need of enhancing memory and / or cognitive function, comprising as an active ingredient a Crtac1B protein, a vector capable of expressing the Crtac1B protein, or a cell expressing the Crtac1B protein, wherein the patient is a patient having amnesia or forgetfulness (excluding the elderly), or a patient with Alzheimer's disease.

3. The agent according to claim 2, wherein the patient is a patient with Alzheimer's disease.

4. An agent for inhibiting the production of amyloid-beta protein in a patient, comprising as an active ingredient a Crtac1B protein, a vector capable of expressing the Crtac1B protein, or a cell expressing the Crtac1B protein.

5. The agent according to claim 4, wherein the patient is a patient with dementia or a patient having an accumulation of Aβ protein in the brain.

6. The agent according to claim 5, wherein the patient with dementia is a patient with Alzheimer's disease.

7. An agent for promoting the clearance of amyloid-beta protein in a patient, comprising as an active ingredient a Crtac1B protein, a vector capable of expressing the Crtac1B protein, or a cell expressing the Crtac1B protein.

8. The agent according to claim 7, wherein the patient is a patient with dementia or a patient having an accumulation of Aβ protein in the brain.

9. The agent according to claim 8, wherein the patient with dementia is a patient with Alzheimer's disease.

10. The agent according to any one of claims 1 to 9, wherein the Crtac1B protein is a polypeptide having an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO:

2.

11. The agent according to any one of claims 1 to 9, wherein the Crtac1B protein is a membrane-bound Crtac1B protein having the amino acid sequence shown in SEQ ID NO: 2, or a soluble Crtac1B protein having the amino acid sequence shown in SEQ ID NO:

5.

12. The agent according to any one of claims 1 to 9, wherein the cell expressing the Crtac1B protein is a neural stem / progenitor cell, an excitatory neuron, or an oligodendrocyte progenitor cell.

13. The agent according to claim 10, wherein the cells expressing the Crtac1B protein are neural stem / progenitor cells, excitatory neurons or oligodendrocyte progenitor cells.

14. The agent according to claim 11, wherein the cells expressing the Crtac1B protein are neural stem / progenitor cells, excitatory neurons or oligodendrocyte progenitor cells.

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