JP2022035562A - Agent for extending axons - Google Patents

Abstract

To provide an agent or a composition useful for, for example, extending axons of nerve cells.SOLUTION: An agent or a composition comprises a component (A) expressing SPARC protein or promoting the expression of SPARC protein (excluding diosgenins).SELECTED DRAWING: None

Description

The present invention relates to an agent or composition capable of extending axons of nerve cells.

In neurodegenerative diseases such as Alzheimer's disease, it is said that axons of nerve cells (hereinafter, also simply referred to as "axons") are atrophied. Axon atrophy makes it difficult to send neurotransmitters through axons to other nerve cells.

All of the current approved drugs for Alzheimer's disease have the effect of enhancing the action of neurotransmitters and do not re-extend atrophied axons. It cannot be improved.

Non-Patent Document 1 and Non-Patent Document 2 report that intraperitoneal administration of diosgenin to mice was observed to enhance memory in normal mice or Alzheimer's disease model mice.

On the other hand, Patent Document 1 describes that neurites are extended by adding SPARC (Secret Protein Acid and Rich in Cystheine) protein to a medium containing nerve cells.

Japanese Patent Publication No. 2005-521427

SCIENTIFIC REPORTS, Volume 2, Number 535, pp1-11. Tohda C, Lee YA, Goto Y, Nemere I. Sci Rep. 2013 Dec 2; 3: 3395. Pp1-8.

An object of the present invention is to provide an agent or composition capable of extending axons of nerve cells.

Another object of the present invention is to provide an agent or composition useful for treating and / or ameliorating a disease associated with axonal dysfunction of nerve cells, and improving and / or maintaining memory.

We have surprisingly found that in atrophied axons, certain proteins (SPARC proteins) are reduced.
Then, the present inventors have found that by expressing (increasing, increasing) the SPARC protein in a nerve cell (in a nerve cell), the atrophied axon re-elongates.

We also focus on the direction in which the nerve cell axons extend, and according to a particular component, the nerve cell axons move toward the projection destination region (eg, the hippocampal nerve cell axis). In the case of axon, it was found that it can be extended (reached) toward the frontal prefield region, which is the projection destination, and further diligent research was carried out to complete the present invention.

Although Non-Patent Documents 1 and 2 describe the extension of axons, it is considered that they extend in various directions instead of in a specific direction, so that there is no idea that they can be extended in a specific direction. rice field.

Further, in Patent Document 1, nerve cells are added to a medium containing a SPARC protein. Considering the size of the SPARC protein, it is highly unlikely that the SPARC protein is incorporated into nerve cells at the time of the addition. Therefore, what can be recognized from Patent Document 1 is that the SPARC protein stimulates some signal that extends the neurite by some action from outside the nerve cell. The idea of expressing the SPARC protein itself in nerve cells cannot be recalled from Patent Document 1.

That is, the present invention relates to the following inventions and the like.
[1]
An agent or composition for axonal extension of nerve cells, which comprises a component (A) that expresses or promotes the expression of SPARC protein (however, component (A) excludes diosgenins).
[2]
Axon of a nerve cell containing a component (A) that expresses a SPARC protein or promotes the expression of a SPARC protein is projected into a projection destination region (a region reached when the target axon is normally projected). Agent or composition for spreading to.
[3]
The agent or composition according to [2], wherein the axon is the axon of a hippocampal nerve cell and the target region is the prefrontal cortex.
[4]
An agent or composition for use in the following uses (1) and / or (2), which comprises a component (A) that expresses or promotes the expression of a SPARC protein (provided that the component (A) is , Excluding diosgenins).
(1) Treatment and / or improvement of diseases associated with axonal dysfunction of nerve cells (2) Improvement and / or maintenance of memory [5]
The agent or composition according to [4], wherein the disease associated with neuronal axonal dysfunction is one or more selected from Alzheimer's disease, spinal cord injury, cerebral contusion, Parkinson's disease and dementia.
[6]
The agent or composition according to any one of [1] to [5], wherein the component (A) contains one or more components selected from the following (A-1) to (A-4).
(A-1) Gene encoding SPARC protein (A-2) One or more enzymes selected from PI3 kinase, MEK1, protein kinase A and protein kinase C (A-3) The enzyme of (A-2) above. Proteins or peptides that activate one or more in nerve cells, vitamins that activate one or more of the above (A-2) enzymes in nerve cells, and one or more of the above (A-2) enzymes in nerve cells. Transcription of one or more enzyme-activating components (A-4) selected from growth factors or growth factors that are activated in the above and steroids that activate one or more of the above-mentioned enzymes (A-2) in nerve cells. One or more transcription activation components selected from a component that activates transcription via the factor c-Jun and a component that activates transcription via the transcription factor c-Jun and the transcription factor Fra1 [7].
The agent or composition according to any one of [2] to [3] and [6], wherein the component (A) contains diosgenins.
[8]
A method for screening an axonal extender for nerve cells, which comprises selecting a component that promotes the expression of a SPARC protein.
[9]
A method of screening an agent for extending axons of nerve cells to a projection destination region, which comprises selecting a component that promotes expression of a SPARC protein.
[10]
A method for screening an agent for use in the following uses (1) and / or (2), which comprises selecting a component that promotes the expression of a SPARC protein.
(1) Treatment and / or improvement of diseases associated with neuronal axonal dysfunction (2) Improvement and / or maintenance of memory

The present invention also includes the following inventions.
[11]
A method for axonal extension of a nerve cell, which expresses (increases) the SPARC protein in the nerve cell (in the nerve cell).
[12]
A method of expressing (increasing) a SPARC protein in a nerve cell (in a nerve cell) and extending the axon of the nerve cell to a projection destination region.
[13]
A method for axonal extension of nerve cells, wherein a component (A) that expresses or promotes the expression of SPARC protein is administered to animals including humans (however, component (A) excludes diosgenins).
[14]
A method for extending axons of nerve cells to a projection destination region by administering a component (A) that expresses or promotes the expression of SPARC protein to animals including humans.
[15]
The method of (1) and / or (2) below, wherein the component (A) that expresses or promotes the expression of the SPARC protein is administered to an animal including a human.
(1) Treatment and / or improvement of diseases associated with neuronal axonal dysfunction (2) Improvement and / or maintenance of memory [16]
Use of a component (A) that expresses or promotes the expression of a SPARC protein for axonal extension of nerve cells (however, the component (A) excludes diosgenins).
[17]
Use of a component (A) that expresses or promotes the expression of SPARC proteins to extend the axons of nerve cells to the projection destination region.
[18]
Use of the component (A) that expresses or promotes the expression of SPARC protein for the following uses (1) and / or (2).
(1) Treatment and / or improvement of diseases associated with neuronal axonal dysfunction (2) Improvement and / or maintenance of memory [19]
A component (A) that expresses a SPARC protein or promotes the expression of a SPARC protein for axonal extension of nerve cells (however, the component (A) excludes diosgenins).
[20]
A component (A) that expresses a SPARC protein or promotes the expression of a SPARC protein for extending axons of nerve cells to a projection destination region.
[21]
A component (A) that expresses a SPARC protein or promotes the expression of a SPARC protein for use in the following uses (1) and / or (2) (however, the component (A) excludes diosgenins). ..
(1) Treatment and / or improvement of diseases associated with neuronal axonal dysfunction (2) Improvement and / or maintenance of memory

According to the present invention, the axons of nerve cells can be extended. In particular, according to the present invention, nerve cell axons can be extended to the projection destination region.
Further, according to the present invention, it is possible to provide an agent or composition useful for treating and / or improving a disease associated with axon dysfunction of nerve cells, and improving and / or maintaining memory.

FIG. 1 shows the result of the number of nerve cells positive only for tracer 2 in hippocampus CA1 in Example 1. FIG. 2 shows the result of the number of nerve cells positive only for tracer 2 in hippocampus CA3 in Example 1. FIG. 3 shows the results of comparison between the SPARC protein expression level and the GAPDH protein expression level in Test Example 1. FIG. 4 shows the expression level of SPARC protein in Example 2. FIG. 5 shows images of pNF-H-positive axons of nerve cells in Example 2. FIG. 6 shows the length of pNF-H positive axons in Example 2. FIG. 7 shows the results of the object recognition memory test in Example 3. FIG. 8 shows the results of the spatial cognitive memory test in Example 3. FIG. 9 shows the results of the number of nerve cells positive only for tracer 2 in hippocampus CA1 in Example 4. FIG. 10 shows the result of the number of nerve cells positive only for tracer 1 in hippocampus CA1 in Example 4. FIG. 11 shows the results of the number of nerve cells positive for both tracers 1 and 2 in hippocampus CA1 in Example 4. FIG. 12 shows the results of the number of BDA-positive axons in hippocampal CA1 in Example 5. FIG. 13 shows the expression level of SPARC protein per axon in Test Example 2. FIG. 14 shows the number of neurons (tracer 1 positive) axon-projected from hippocampus CA1 to the frontal prefield before AAV injection in Example 6 (FIG. 14A) and from hippocampus CA1 to frontal prefield after AAV injection. The number of neurons (tracer 2 positive) that were axon-projected (FIG. 14B) is shown in. FIG. 15 shows the number of neurons (tracer 1 positive) axon-projected from hippocampus CA3 to the frontal prefield before AAV injection in Example 7 (FIG. 15A) and from hippocampus CA3 to frontal prefield after AAV injection. The number of neurons (tracer 2 positive) that were axon-projected (FIG. 15B) is shown in.

[Agent or composition]
The agent or composition of the present invention contains a component (A) that expresses a SPARC protein or promotes the expression of a SPARC protein, and can be used in various applications. The agent or composition of the present invention may contain one or more components (A).

[Use]
The agent or composition of the first aspect is used for axonal extension of nerve cells. In the agent or composition of the first aspect, the component (A) usually does not contain diosgenins (for example, diosgenin, diosgenin derivatives, pharmaceutically acceptable salts thereof, etc.).

The agent or composition of the second aspect is used to extend the axons of nerve cells to the area to which they are projected. That is, the agent or composition of the second aspect directs the axon of the nerve cell toward the projection destination region among the uses of the agent or composition of the first aspect (use of extending the axon of the nerve cell). It is used for a more limited purpose of extending (in a direction to reach).
In the agent or composition of the second aspect, at least a part of the axon may be extended toward the projection destination region, and the axon may be in a direction other than the projection destination region (regardless of the directionality). It does not exclude the case of extending to.
For example, in the agent or composition of the second aspect, 70% or more (for example, 80% or more, 90% or more, 95% or more, etc.) of the atrophied axons are directed toward the projection destination region. It may be extended.
The ratio of axons extending toward the projection destination region among the atrophied axons may be determined by, for example, image analysis using a tracer such as the method described in the embodiment. For example, nerve cells (a) that are not labeled with a tracer prior to administration of the agent or composition of the second aspect indicate nerve cells with axon atrophy and after administration of the agent or composition of the second aspect. The injected tracer-labeled nerve cell (b) indicates a nerve cell in which an axon is extended to the projection destination region. By comparing the number of cells (a) with the number of cells (b), it is possible to confirm the proportion of atrophied axons extending toward the projection destination region.

The agent or composition of the second aspect may be used, for example, for extending the axons of hippocampal neurons toward the prefrontal cortex (or prefrontal cortex region). In normal neurons, the axons of hippocampal neurons extend toward the prefrontal area in circuits involved in cognitive function. Therefore, the agent or composition of the second aspect used for such applications can further contribute to the construction of normal neural circuits involved in cognitive function.

Since the agents or compositions of the first and second aspects can extend axons (further, extend toward the projection destination area), a symptom or disease suggesting a relationship with axon atrophy. It can be applied to applications such as treatment and improvement (for example, (1) treatment and / or improvement of diseases associated with axon dysfunction of nerve cells, which will be described later).
In the agents or compositions of the first and second aspects, the type of nerve cell is not particularly limited, and the axon may be a nerve cell axon corresponding to the disease or symptom to be treated or improved. Further, in the agent or composition of the second aspect, the projection destination region may be a region reached when the axon of the corresponding nerve cell is normally projected.
For example, in the case of a disease or symptom involving hippocampal neurons such as Alzheimer's disease, the axons of the hippocampal neurons may be extended (further, extended toward the prefrontal area). In addition, the agents or compositions of the first and second aspects can be applied to (2) improvement and / or maintenance of memory when the axons of hippocampal neurons are extended.

The agent or composition of the third aspect is useful for the following uses (1) and / or (2). In the agent or composition of the third aspect, the component (A) usually does not contain diosgenins (for example, diosgenin, diosgenin derivatives, pharmaceutically acceptable salts thereof, etc.).
(1) Treatment and / or improvement of diseases associated with nerve cell axon dysfunction (2) Improvement and / or maintenance of memory ability The agent or composition of such a third aspect is a nerve axon. May be accompanied by extension of the nerve axon (first aspect) or extension of the nerve axon toward the projection destination area (second aspect).

Diseases associated with axon dysfunction of nerve cells are not particularly limited, and examples thereof include spinal cord injury, cerebral contusion, Alzheimer's disease (AD), Parkinson's disease, and dementia. In the present invention, the dementia does not include Alzheimer's disease (Alzheimer's disease), but includes cerebrovascular dementia, Lewy body dementia, frontotemporal dementia, Pick disease and the like. Among these diseases, AD, spinal cord injury, cerebral contusion, Parkinson's disease, dementia and the like are particularly preferable.

The agent or composition of the present invention (the agent or composition of the first, second and third aspects) can improve and / or maintain memory, regardless of whether or not it leads to any disease / symptom.

For example, the agent or composition of the present invention may be used to (I) improve and / or maintain the memory of a subject (human) suffering from a specific disease / symptom as described in (1) above. II) It may be used to improve and / or maintain the memory of a subject (human) who has a disease / symptom other than the above (1), and (III) the memory of a subject (human) who does not have any disease / symptom. It may be used for improvement and / or maintenance (for example, improvement or improvement of memory loss due to aging or the like).

The agent or composition of the present invention may improve various symptoms or diseases as described above, and such symptoms or diseases may be those in the chronic phase (chronic disease, chronic symptoms). The chronic phase is not particularly limited and depends on the symptom or disease, but usually refers to the condition after the lapse of the variable phase (acute phase, severe symptom).

Usually, it is difficult to treat or improve the symptoms in the chronic phase, but according to the agent or composition of the present invention, even a disease or symptom in the chronic phase can be efficiently treated and / or ameliorated.

Further, since the agent or composition of the present invention can extend not only atrophied axons but also non-atrophied axons, it is possible to prevent specific diseases / symptoms as described in (1) above.

[Ingredient (A)]
Examples of the component (A) include a component capable of expressing (increasing) the SPARC protein in a nerve cell or contributing to the expression (increasing). The expression of the SPARC protein may be in a nerve cell (in a nerve cell), may be in the axon of the nerve cell, or may be in a site other than the axon. When expressed in axons, it may be atrophied axons or non-atrophied axons.
Typically, component (A) is a component that expresses a SPARC protein (or produces a SPARC protein or expresses a SPARC gene) or promotes (or stimulates) expression of a SPARC protein. It should be.

The nucleotide sequence of the SPARC gene and the amino acid sequence of the SPARC protein can be easily obtained from a known database (DDBJ / GenBank / EMBL, etc.). For example, the accession numbers of the base sequences of the SPARC genes of humans, mice, and rats and the amino acid sequences of the SPARC proteins are as follows.
Human: (BC072457 etc.) (base sequence), (CAG33080) (amino acid sequence)
Mice: (BC004638 etc.) (base sequence), (CAJ18514) (amino acid sequence)
Rats: (BC061777, etc.) (base sequence), (XP_032770322) (amino acid sequence)

Examples of the component expressing the SPARC protein include a gene encoding the SPARC protein. Such genes are typically SPARC genes. Further, such a gene may be a gene having sequence homology of, for example, 70% or more (for example, 80% or more) with the SPARC gene.

Examples of the component that promotes the expression of the SPARC protein include (A-2) PI3 kinase (PI3K, Phosphoinoside 3-kinase), MEK1 (mitogen-activated protein kinase 1), protein kinase A (PKA), and protein kinase C (PKA). A component that activates one or more of enzymes selected from PI3 kinase, MEK1, protein kinase A, and protein kinase C in enzymes such as PKC) and (A-3) nerve cells (enzyme activation component) [for example, Proteins or peptides that activate one or more of the enzymes (eg, peptide hormones such as insulin, leptin, proteins such as bone-forming protein 1, etc.), vitamins that activate one or more of the enzymes (eg,). (Retinoic acid, etc.), a growth factor or growth factor that activates one or more of the enzymes (for example, transforming growth factor β1, a kinase-derived growth factor, an insulin-like growth factor 1, etc.), and one or more of the enzymes are activated. Kinases [for example, diosgenins (eg, diosgenins, diosgenin derivatives, pharmaceutically acceptable salts thereof, etc.), etc.], (A-4) A component that activates transcription mediated by the transcription factor c-Jun. , Kinase-activating components such as components that activate transcription via the transcription factor c-Jun and the transcription factor Fra1.
When the transcription factor described in (A-4) binds to the promoter region of the SPARC gene, it can promote transcription from the SPARC gene to mRNA, and thus can promote the expression of the SPARC protein.

で表わされるステロイドサポゲニンであり、サンヤク（Dioscorea rhizome）、及びトリゴネラ属（Trigonella spp.）、アマドコロ属（Polygonatum spp.）、シオデ属（Smilax spp.）のようなハーブ薬等いくつかの種の植物に含まれていることが知られている。 Diosgenin has the following chemical formula (I)

Is a steroid sapogenin represented by, and of several species such as dioscorea rhizome, and herbal medicines such as Trigonella spp., Solomonatum spp., And Smilax spp. It is known to be contained in plants.

In the present invention, a diosgenin derivative usually refers to a compound that can be an equivalent of diosgenin.
The diosgenin and diosgenin derivatives are not particularly limited, and may be commercially available products, may be manufactured according to a known method, a known method itself, or a method similar thereto, and are extracts from natural products. May be good.
For example, a diosgenin derivative may be an equivalent that can be achieved by chemical modification such as introducing a substituent into diosgenin or converting a substituent, and is a diosgenin glycoside (such as diosin) extracted from a natural product. May be.

The diosgenin derivative is not particularly limited, but is, for example, a compound in which the hydroxyl group at the C3 position of diosgenin is substituted, a compound having a substituent at the C2-position (or a compound in which the hydrogen atom at the C2-position is substituted), or a compound at the C4 position. Specific examples thereof include a compound having a group (or a compound in which the hydrogen atom at the C4 position is substituted), a compound having a substituent at the C6 position (or a compound in which the hydrogen atom at the C6 position is substituted), or a salt thereof. Examples thereof include an ester derivative of the hydroxyl group at the C3 position (for example, an amino acid derivative, an aminosulfonic acid derivative, a carbamate derivative), a halogenated derivative of the hydroxyl group at the C3 position, and the like.

（式中、Ｒ^１、Ｒ^２、Ｒ^３及びＲ^４は、同一又は異なって水素原子又は置換基を示す。ただし、Ｒ^２、Ｒ^３及びＲ^４が水素原子であるとき、Ｒ^１はヒドロキシル基でない。） Examples of the diosgenin derivative (and its salt) include a compound represented by the following formula (I-1) and a salt thereof (a pharmaceutically acceptable salt).

(In the formula, R ¹ , R ² , R ³ and R ⁴ indicate the same or different hydrogen atom or substituent. However, when R ² , R ³ and R ⁴ are hydrogen atoms, R ¹ is hydroxyl. Not a group.)

In ^R1 , the substituent is a hydrocarbon group {for example, an alkyl group [for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, t-butyl group. A linear or branched alkyl group such as a group, a pentyl group (eg, a C _1-12 alkyl group, preferably a C _1-8 alkyl group)], a cycloalkyl group (eg, a cyclopentyl group, a cyclohexyl group, a cycloheptyl). C _4-10 cycloalkyl group such as group, cyclooctyl group, preferably C _5-8 cycloalkyl group), aralkyl group (for example, C _6-10 aryl C _1-4 alkyl group such as benzyl group and phenethyl group). , Saturated or unsaturated aliphatic hydrocarbon groups such as polycyclic aliphatic hydrocarbon groups (eg, decalinyl group, norbornyl group, adamantyl group, dimethyladamantyl group, etc.); aryl groups (eg, phenyl group, trill group, xylyl group). Aromatic hydrocarbon groups such as C _6-10 aryl groups such as groups}, groups containing hetero (nitrogen atoms, oxygen atoms, sulfur atoms, phosphorus atoms, etc.) {eg, oxygen atom-containing groups [eg, hydroxyl groups, Oxo group (= O), group-OR ^a , group-O-CO-R ^a , group-O-CO-N (R ^b ) ₂ , group-O-CO-OR ^a , group-O-CO -S-R ^a , group-OR ^c , group-O-SO ₂ -OH, group-O-PO ₂ -OH, group- (OR ^d ) _k -R ^e , carboxyl group, group-CO-OR ^a , group-CO-N (R ^b ) ₂ , etc.], nitrogen atom-containing group [eg, amino group, group-NR ^a R ^b , group-NR ^b -CO-OR ^a , group-NR ^b -CO -N (R ^b ) ₂ , nitrogen-containing ring group (eg, group corresponding to pyridine, pyrrolin, pyrrol, indol, etc.), etc.], sulfur atom-containing group [eg, mercapto group, group-SR ^a , group-S- S-R ^a , sulfo group (-SO ₃ H), group-SO ₂ -R ^b , group-S-CO-N (R ^b ) ₂ , etc.], phosphorus atom-containing group [eg, phosphate group (H ₂ ). PO _4- ), group-PO _3H , etc.], amino acid group [or amino acid residue, for example, the hydroxyl group at the 3-position (corresponding to the substitution position of R ¹ in the above formula) constituting diosgenin, and the amino acid (glycine). , A group formed by an ester bond with a carboxyl group of alanine, etc.], etc.}, a halogen atom (eg, a fluorine atom, a chlorine atom, a bromine source) Child, iodine atom, etc.).
In the above formula, Ra is a hydrocarbon group (for example, the above-exemplified hydrocarbon group such as an alkyl group), and R ^b is a hydrogen atom or a hydrocarbon group (for example, the above-exemplified hydrocarbon group such as an alkyl group ⁾ . ), R ^c is a sugar (or a sugar chain or a residue of a sugar), R ^d is an alkylene group (for example, a C _2-4 alkylene group such as an ethylene group, a propylene group or a trimethylene group), and ^Re is a hydrogen atom or a hydroxyl group. A group or a hydrocarbon group (for example, the above-exemplified hydrocarbon group such as an alkyl group (such as a methyl group)) and k indicate an integer of 2 or more (for example, 2 to 10 ^{), respectively, and Ra and R b are} the same. Alternatively, they may be different groups, and when there are a plurality of R ^bs , they may be the same or different.

In R ^a and R ^b , the hydrocarbon group (such as an alkyl group) may further have a substituent. The substituent is not particularly limited, but includes the above-exemplified substituent, for example, an oxygen atom-containing group (for example, a hydroxyl group, a carboxyl group, a group-OR ^a , a group-O-CO-R ^a , etc.) and a nitrogen atom. Examples include a group (eg, amino group, group-NR ^a R ^b ), a sulfur atom-containing group (eg, mercapto group, group-SR ^a , sulfo group, group-SO ₂ -R ^b , etc.).
The hydrocarbon group may have these substituents alone or in combination of two or more.
When the hydrocarbon group has a substituent, the number of substituents may be 1 or more, for example, 1 to 10 (for example, 1 to 8), preferably 1 to 6 (for example, 1 to 4), and further. It may be preferably about 1 to 3.

When R ¹ is a substituent, a typical R ¹ may be, for example, a hydrocarbon group [eg, an alkyl group (eg, a group- (CH ₂ ) _n -CH ₃ ), a cycloalkyl group, an aralkyl group, etc.]. , Heteroatomic group {eg, oxygen atom-containing group [eg, hydroxyl group, group-O- (CH ₂ ) _n -CH ₃ , group-O- (CH ₂ ) _m -NH ₂ , group-O- (CH) ₂ ) _m -COOH, group-O- (CH ₂ ) _m -SO ₃ H, group-O-CO- (CH ₂ ) _n -CH ₃ , group-O-CO-NH- (CH ₂ ) _n -CH ₃ , Group-O-CO-NR- (CH ₂ ) _n -CH ₃ , Group-O-CO-NH-CH (R ^b ) -COOH, Group-O- (CH ₂ ) _n -CO-NH-AD (In the formula, AD indicates an adamantyl group (1-adamantyl group, 2,6-dimethyladamantan-1-yl group, etc.)), group-O-CO-NH- (CH ₂ ) _m -SO ₃ H, group. -O-CO-NH- (CH ₂ ) _m -COOH, group -O-CO-O- (CH ₂ ) _n -CH ₃ , group -O-CO-S- (CH ₂ ) _n -CH ₃ , group -O-SU (in the formula, SU indicates a sugar chain), group-O-SO ₂ -OH, group-O-PO ₂ -OH, group- (OCH ₂ CH ₂ ) _m -CH ₃ , group- (OCH ₂ CH ₂ CH ₂ ) _m -CH ₃ , carboxyl group, group-COO (CH ₂ ) _n CH ₃ , group-CO-NH- (CH ₂ ) _n -CH ₃ , group-SO ₃ H, group- SO _2- (CH ₂ ) _n -CH ₃ , group-SO ₂ -Ph (in the formula, Ph indicates a phenyl group), group-CO-NH-CH (R ^b ) -COOH, group-CO-NH -(CH ₂ ) _n -SO _3H , etc.], nitrogen atom-containing group [eg, amino group, group-NH- (CH ₂ ) _n -CH ₃ , group-NH- (CH ₂ ) _n -NH _2, group -NH-CH (R ^b ) -COOH, group-NH- (CH ₂ ) _m -SO ₃ H, group-NH- (CH ₂ ) _m -SO ₂ H, group -NH-CO-O- (CH ₂ ) ) _N -CH ₃ , group-NH-CO-NH ₂ , group-NH-CO-NH-AD (in the formula, AD is an adamantyl group (1-adamantyl group, 2,6-dimethyladamantan-1-yl group, etc.) ), Group-NH-CO-NH-C H (R ^b ) -COOH, group-NH-CO-NH- (CH ₂ ) _m -SO ₃ H, group-NH-CO-NH- (CH ₂ ) _m -COOH, etc.], sulfur atom-containing group [eg , Mercapto group, group-S- (CH ₂ ) _n -CH ₃ , group-S- (CH ₂ ) _m -COOH, group-S- (CH ₂ ) _m -CH (NH ₂ ) -COOH, group-S -CO-NH-AD (in the formula, AD indicates an adamantyl group (1-adamantyl group, 2,6-dimethyladamantan-1-yl group, etc.)), group-SS- (CH ₂ ) _m -CH (NH ₂ ) -COOH, group-SO ₃ H, etc.], phosphorus atom-containing group [eg, group-PO ₃ H, etc.], amino acid group (eg, group-O-CO-CH ₂ -NH ₂ etc.)} , Halogen atoms (eg, fluorine atom, chlorine atom, bromine atom, iodine atom, etc.) and the like.
In the above formula, m is an integer of 1 or more (for example, 1 to 10, preferably 1 to 4, more preferably 1 or 2), and n is an integer of 0 or more (for example, 0 to 10, preferably 0 to 0 to 2). 7), where R ^b is the same as above [ie, a hydrogen atom or a hydrocarbon group (eg, an alkyl group, etc.)].

^{In R2} , R3 and ^R4 , examples of the substituent include the same substituents as those exemplified in the section of ^R1 .
When R ² and / or R ⁴ are substituents, typical substituents include an oxygen atom-containing group, a nitrogen atom-containing group, a sulfur atom-containing group, an amino acid group, a halogen atom and the like.
When R ³ is a substituent, a typical substituent includes a halogen atom and the like.

In the formula (I-1), the combinations of R ¹ to R ⁴ are not limited, and all combinations are included. Typical combinations of R ¹ to R ⁴ include, for example, the following combinations.
(1) A combination in which R ¹ is a substituent other than a hydroxyl group and R ² to R ⁴ are hydrogen atoms (2) R ¹ is a substituent other than a hydroxyl group, R ² is a substituent, and R ³ and R ⁴ are hydrogen. Combinations that are atoms (3) Combinations where R ¹ is a substituent other than a hydroxyl group, R ³ is a substituent, and R ² and R ⁴ are hydrogen atoms (4) R ¹ is a substituent other than a hydroxyl group, R ² and A combination in which R ³ is a substituent and R ⁴ is a hydrogen atom (5) A combination in which R ¹ is a substituent other than a hydroxyl group, and R ² , R ³ and R ⁴ are substituents (6) R ¹ is a non-hydroxyl group Substituents, R ² and R ³ are hydrogen atoms, R ⁴ is a substituent (7) R ¹ is a substituent other than a hydroxyl group, R ³ is a hydrogen atom, and R ² and R ⁴ are substituents. Combination (8) Combination in which R ¹ is a substituent other than the hydroxyl group, R ² is a hydrogen atom, and R ³ and R ⁴ are substituents. (9) R ¹ is a hydroxyl group, R ² is a substituent, R ³ and R A combination in which ⁴ is a hydrogen atom (10) A combination in which R ¹ is a hydroxyl group, R ³ is a substituent, and R ² and R ⁴ are hydrogen atoms (11) R ¹ is a hydroxyl group, and R ² and R ³ are substituents. , R ⁴ is a hydrogen atom (12) R ¹ is a hydroxyl group, R ² to R ⁴ are substituents (13) R ¹ is a hydroxyl group, R ² and R ³ are hydrogen atoms, R ⁴ is Substituent combination (14) R ¹ is a hydroxyl group, R ² is a hydrogen atom, R ³ and R ⁴ are substituents (15) R ¹ is a hydroxyl group, R ³ is a hydrogen atom, R ² and R Combination in which ⁴ is a substituent

で表わされる（３β，２５Ｒ）－３－（２－アミノエタノイロキシ）－スピロスト－５－エン｛(3β, 25R)-3-(2-Aminoethanoyloxy)-spirost-5-ene｝、及びその薬学的に許容される塩、又は下記化学式（ＩＩＩ）

で表わされる（３β，２５Ｒ）－３－フルオロスピロスト－エン｛(3β, 25R)-3-Fluorospirost-5-ene｝、並びに、（３β，２５Ｒ）－３－（２－アミノエチルスルホニルオキシ）－スピロスト－５－エン｛(3β, 25R)-3-(2-Aminoethylsulfonyl-oxy)-spirost-5-ene｝、（３β，２５Ｒ）－３－（２－アミノプロピルスルホニルオキシ）－スピロスト－５－エン、（３β，２５Ｒ）－３－[Ｎ－（２，６－ジメチルアダマンタン－１－イル）カルバモイルオキシ]－スピロスト－５－エン、（３β，２５Ｒ）－３－｛［Ｎ－（２，６－ジメチルアダマンタン－１－イル）カルバモイル］アミノ｝－スピロスト－５－エン、（３β，２５Ｒ）－３－［Ｎ－（２，６－ジメチルアダマンタン－１－イル）カルバモイルチオ］－スピロスト－５－エン、（３β，２５Ｒ）－３－｛［Ｎ－（アダマンタン－１－イル）カルバモイル］アミノ｝－スピロスト－５－エン、（３β，２５Ｒ）－３－［Ｎ－（アダマンタン－１－イル）カルバモイルチオ］－スピロスト－５－エン、及び（３β，２５Ｒ）－３－［Ｎ－（アダマンタン－１－イル）カルバモイルオキシ］－スピロスト－５－エン[又は（３β，２５Ｒ）－３－（１－アダマンチル－アミノカルボニルオキシ）－スピロスト－５－エン｛(3β, 25R)-3-(1-adamantyl-aminocarbonyloxy)-spirost-5-ene｝]等が挙げられる。 The diosgenin derivative is not particularly limited, but specifically, for example, the following formula (II).

(3β, 25R) -3- (2-aminoethanoyloxy) -spirost-5-ene {(3β, 25R) -3- (2-Aminoethanoyloxy) -spirost-5-ene}, and its pharmacy. Acceptable salt, or the following chemical formula (III)

(3β, 25R) -3-fluorospirost-ene {(3β, 25R) -3-Fluorospirost-5-ene}, and (3β, 25R) -3- (2-aminoethylsulfonyloxy). -Spirost-5-ene {(3β, 25R) -3- (2-Aminoethylsulfonyl-oxy) -spirost-5-ene}, (3β, 25R) -3- (2-Aminopropylsulfonyloxy) -Spirost-5 -En, (3β, 25R) -3- [N- (2,6-dimethyladamantane-1-yl) carbamoyloxy] -spirost-5-ene, (3β, 25R) -3-{[N- (2) , 6-dimethyladamantane-1-yl) carbamoyl] amino} -spirost-5-ene, (3β, 25R) -3- [N- (2,6-dimethyladamantan-1-yl) carbamoylthio] -spirost- 5-ene, (3β, 25R) -3-{[N- (adamantane-1-yl) carbamoyl] amino} -spirost-5-ene, (3β, 25R) -3- [N- (adamantane-1-) Il) Carbamoylthio] -spirost-5-ene, and (3β, 25R) -3- [N- (adamantan-1-yl) carbamoyloxy] -spirost-5-ene [or (3β, 25R) -3- (1-adamantyl-aminocarbonyloxy) -spirost-5-ene {(3β, 25R) -3- (1-adamantyl-aminocarbonyloxy) -spirost-5-ene}] and the like can be mentioned.

As the diosgenin derivative (or a salt thereof), a commercially available product may be used, or a product synthesized by a known method may be used. For example, when a substituent is introduced into R ¹ , various substituents can be introduced via the hydroxyl group originally possessed by diosgenin (the hydroxyl group at the 3-position). When a halogen atom is introduced into R ² or R ³ , R ¹ is substituted (oxidized) with an oxo group, the adjacent carbon of the generated ketone is halogenated, and R ² or R ³ is converted to halogen (further). If necessary, a method of converting (reducing) the oxo group to a hydroxyl group can be adopted. Further, when a halogen atom is introduced as R ⁴ , it can be introduced via electrophilic halogenation or the like for an unsaturated bond. Furthermore, it is also possible to introduce various substituents by using a nucleophile containing a hetero atom (oxygen atom, nitrogen atom, sulfur atom, etc.).

In the present invention, the "pharmaceutically acceptable salt" (or "salt") includes, and is not particularly limited, those that form a pharmaceutically acceptable salt with diosgenin or the like. Specifically, for example, hydrohalogenates (for example, fluorinated hydrochloride, hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid salts (for example, sulfates, nitrates, perchlorates, etc.) , Phosphates, carbonates, bicarbonates, etc.), organic carboxylates (eg, acetates, oxalates, maleates, tartrates, fumarates, citrates, etc.), organic sulfonates (eg, acetates) Methane sulfonate, trifluoromethane sulfonate, ethane sulfonate, benzene sulfonate, toluene sulfonate, camphor sulfonate, etc.), amino acid salts (eg, asparagate, glutamate, etc.), organic amine salts (eg, asparagate, glutamate, etc.) For example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picolin, dicyclohexylamine, N, N'-dibenzylethylenediamine, arginine and lysine), quaternary amine salts, alkali metal salts (eg sodium salt, potassium salt, etc.) ), Alkaline earth metal salts (for example, magnesium salts, calcium salts, etc.) and the like.

[Form / administration]
The form of the agent or composition of the present invention is not particularly limited, and the component (A) may be used as it is as an agent (for example, a powder) or a composition, together with other components (for example, a carrier, an excipient, etc.). It may be formulated.

Examples of other components include components generally used as raw materials for pharmaceutical products, and are not particularly limited. For example, carriers, excipients, binders, disintegrants, lubricants, coating agents, coloring agents, and flavoring agents. Examples thereof include agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants and the like. These other components may be used alone or in combination of two or more.

The carrier is not particularly limited, and is, for example, animal and vegetable oils such as soybean oil, beef fat, and synthetic glyceride; hydrocarbons such as liquid paraffin, squalane, and solid paraffin; ester oils such as octyldodecyl myristate and isopropyl myristate; Higher alcohols such as alcohols and behenyl alcohols; Silicon resin; Silicone oils; Polyoxyethylene fatty acid esters, sorbitan fatty acid esters, glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene hydrogenated castor oil, polyoxyethylene polyoxypropylene block copolymers, etc. Surfactants; water-soluble polymers such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymers, polyethylene glycol, polyvinylpyrrolidone, methyl cellulose; lower alcohols such as ethanol, isopropanol; glycerin, propylene glycol, dipropylene glycol, sorbitol, etc. Polyhydric alcohols; sugars such as glucose and sucrose; inorganic powders such as silicic acid anhydride, aluminum magnesium silicate, aluminum silicate; purified water and the like.

Examples of the excipient include lactose, cornstarch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, silicon dioxide and the like.

Examples of the binder include polyvinyl alcohol, gelatin, methyl cellulose, ethyl cellulose, gum arabic, tragant, gelatin, shellac, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyvinyl acetal diethylaminoacetate, corn starch and the like.

Examples of the disintegrant include corn starch, low-substituted hydroxypropyl cellulose, crospovidone, crystalline cellulose, precipitated calcium carbonate, croscarmellose sodium, calcium citrate, dextrin, pectin, calcium carboxymethyl cellulose and the like.

Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, light anhydrous silicic acid, sucrose fatty acid ester, and the like, and examples of the flavoring agent include cocoa powder, menthol, aromatic powder, peppermint oil, and dragon brain. Cinnamon powder is used.

The form (dosage form) of the agent or composition is not particularly limited, and for example, tablets, powders, fine granules, granules, dry syrups, coated tablets, orally disintegrating tablets, chewable tablets, capsules, soft capsules. , Syrups, oral solutions, troches, jellies, inhalants, suppositories, injections, ointments, eye drops, eye ointments, nasal drops, ear drops, paps, lotions, external solutions, sprays Examples thereof include agents, external aerosols, creams, gels, tapes, buccal tablets, sublingual tablets, vaginal suppositories, vaginal tablets, and rectal soft capsules.

The form of administration (administration) of the agent or composition is not particularly limited, and may be oral administration or parenteral administration. Parenteral administration includes, for example, rectal administration, nasal administration, transpulmonary administration, injection administration (for example, intravenous administration, intrathecal administration, intraepidural administration, intramuscular administration, subcutaneous administration, intraperitoneal administration). , Intraarterial administration, intraarterial administration, intracardiac administration, intracapsular administration, intradermal administration, intralesional administration, intraocular administration, intrathoracic administration, epidural administration, intrauterine administration, intracerebroventricular administration, intraparenchymal administration, spinal cord Intravenous administration) and the like.

The agent or composition of the present invention may be preferably used, such as oral administration, intravenous administration, intramuscular administration, and intrathecal administration, although it depends on the type of the component (A).

When component (A) is a gene, component (A) may be included in the agent or composition in the form of a vector into which the gene has been introduced (or loaded with the gene). The method for introducing the gene into the vector is not particularly limited, and a known method may be used.

When an agent or composition containing a vector is administered, the administration form includes, for example, injection administration (for example, intraparenchymal administration, intrathecal administration, intraventricular administration, intravenous administration, intramuscular administration, etc.), intramuscular administration, etc. , Subcutaneous administration and the like may be preferably used.

When the component (A) is a gene or the like, it may be contained in the agent or composition in the form in which the component (A) is introduced (included).
For example, the target cells are taken out from the body of the administration target or the non-administration target by blood sampling, biopsy, etc., cultured in vitro, the gene is introduced into the cells by a vector, and the obtained gene-introduced cells are administered as an agent or composition. It may be (or returned to the body of the subject).

The vector may be a viral vector or a non-viral vector (eg, liposomes, plasmids, macromolecules, etc.). As the vector, a viral vector or the like may be preferably used from the viewpoint of gene introduction efficiency and the like.

Examples of the virus of the virus vector include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus, DNA virus such as SV40, RNA virus and the like. Can be mentioned.

The promoter of the viral vector may be any promoter as long as it promotes specific expression in nerve cells, and examples thereof include a synapsin I promoter and a neuron-specific enolase promoter.

In the agent or composition of the present invention, the amount of the component (A) is not particularly limited and can be appropriately selected depending on the dosage form, dosage and the like.

The dose (intake, dose) of the agent or composition of the present invention depends on the type of component (A), the degree of disease / symptom, age, sex, body weight, administration form, specific type of disease, and the like. However, for example, the amount of the component (A) per day is about 0.01 to 50 mg / kg, preferably about 0.05 to 10 mg / kg, and more preferably about 0.1 to 1 mg / kg. May be good.

When the component (A) is a gene, the dose of the gene is preferably 1 × 10 ¹⁰ to 5 × 10 ¹² vg / human, for example, in the case of intraparenchymal administration to humans. May be about 5 × 10 ¹⁰ to 1 × 10 ¹² vg / human, more preferably 1 × 10 ¹¹ to 5 × 10 ¹¹ vg / human.

Administration may be divided into one or more doses.

The agent or composition of the present invention may be a pharmaceutical product or a pharmaceutical composition, or may be a quasi-drug.

The agent or composition of the present invention can also be provided in the form of a kit, if desired.
The kit may be a kit including a container such as a pack or a dispenser device.
Further, the kit may be composed of a container containing the component (A) and a container containing other components.

Although it depends on the type of the component (A), the agent or composition of the present invention may be in the form of a food or drink. Therefore, the present invention also includes foods and drinks containing the component (A) (or the agent or composition). Further, from the viewpoint of the component (A) (or the agent or composition), the component (A) may be an additive for food and drink.

In such foods and drinks, the preferred embodiment of the component (A) may be the same as described above.

Food and beverages include general foods including so-called health foods, as well as foods with health claims such as foods for specified health use and foods with nutritional claims stipulated in the Health Function Foods System of the Ministry of Health, Labor and Welfare, and supplements ( Dietary supplements), feeds, food additives and the like are also included in the food and drink of the present invention.

In addition, the food and drink is a food and drink for a specific target person [for example, for the elderly, for a patient or a sick person (for example, for a patient having Alzheimer's disease (for example, the above-exemplified disease or symptom)]]. There may be.

In order to use the component (A) in a food and drink, the food and drink can be produced as it is or mixed with various nutritional components and the like as raw materials for the food and drink such as processed meat and soft drinks.

When the ingredient (A) is used as a health food, a dietary supplement, etc., for example, tablets, capsules (soft capsules, hard capsules, etc.), powders, granules, liquids (suspensions, syrups, etc.) are used by conventional means. Agents, etc.), emulsions, jellies, sticks, etc. can be prepared. The tablets also include disintegrating tablets (orally disintegrating tablets).

The food and drink may contain a food additive (food additive). The food additive is not particularly limited, but for example, excipients (eg, wheat starch, corn starch, cellulose, lactose, sucrose, mannitol, sorbitol, xylitol, pregelatinized starch, casein, magnesium silicate aluminate, etc. Calcium silicate, etc.), excipients (eg, pregelatinized starch, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.), disintegrants (eg, cellulose, hydroxypropylcellulose, corn starch, etc.), fluidizers (eg, light anhydrous silicic acid, etc.) , Sucrose fatty acid esters, etc.), oils (eg, soybean oil, sesame oil, olive oil, flaxseed oil, sesame oil, rapeseed oil, coconut oil, corn oil and other vegetable oils or animal / fish-derived oils), nutrients (eg, various Minerals, various vitamins, amino acids), fragrances, sweeteners, flavoring agents, coloring agents, solvents (ethanol), salts, surfactants, pH adjusters, buffers, antioxidants, stabilizers, gelling agents, increase Examples include thickeners, lubricants, encapsulants, suspending agents, coating agents, preservatives and the like.

Food additives may be used alone or in combination of two or more.

When the component (A) is used as a food and drink additive, the food and drink is not particularly limited, but for example, foods [for example, noodles (soba, udon, Chinese noodles, instant noodles, etc.), confectionery (candy, candy, etc.) Gum, chocolate, snacks (potato chips, etc.), biscuits, cookies, gummy, jelly, jam, butter, cream (cream puffs, etc.), cakes, etc.), breads, marine or processed livestock foods (kamaboko, ham, sausage, etc.), Dairy products (processed milk, fermented milk, etc.), fats and oils and processed fats and oils (salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressings, etc.), seasonings (sauce, sauce, etc.), retort foods (curry, stew, etc.) , Bowls, porridge, miscellaneous dishes, etc.), chilled confectionery (ice cream, sherbet, shaved ice, etc.), fried foods (croquette, fried potatoes, fried chicken, etc.)], beverages (tea beverages, soft beverages, carbonated beverages, nutritional beverages, fruits Beverages, lactic acid beverages, etc.).

The blending amount of the component (A) in the above food and drink varies depending on the addition form and the administration form, and can be selected from a wide range. For example, 0.0001% by weight or more (for example, 0.001 to 50% by weight). It may be 0.003% by weight or more (for example, 0.005 to 30% by weight), 0.01% by weight or more (for example, 0.05 to 10% by weight), and the like.

In the food and drink, the intake amount (or dose or dose) of the component (A) can be selected from the same range as described above.

[Screening method]
The present invention also includes screening methods for various agents, including selecting components that promote expression of SPARC proteins.
The methods include (i) a method for screening a nerve cell axon extender, (ii) a method for screening an agent for extending a nerve cell axon to a projection destination region, and (iii) (1) a nerve cell. It may be a method for screening agents for use in the treatment and / or amelioration of diseases associated with axonal dysfunction, and / or (2) improvement and / or maintenance of memory.

In the screening method of the present invention, the test substance is not particularly limited, and is, for example, nucleic acid, peptide, protein, non-peptide compound, synthetic compound, fermentation product, cell extract, cell culture supernatant, plant extract, mammal. It may be animal tissue extract, plasma, or the like. Further, the test substance may be a novel substance or a known substance. The test substance may form a salt. The salt of the test substance may be a salt with a physiologically acceptable acid or base.

The screening method of the present invention includes, for example, a step of contacting a cell with a test substance, a step of measuring the expression level of the SPARC gene in the cell, and an expression level of the SPARC gene in a cell in which the expression level is not brought into contact with the test substance. It may include a step of selecting a test substance for comparison and improving the expression level of the SPARC gene.

The cells used in the screening method may be in vivo cells or cultured cells. The cultured cells may be primary cultured cells or cell lines.
Further, the cell may be a cell having a SPARC gene, or may be a cell in which a SPARC gene is introduced into a cell not having a SPARC gene.
Examples of cells having the SPARC gene include nerve cells, fibroblasts, endothelial cells and the like.
In addition, examples of cells that do not have the SPARC gene include blood cells and the like.

The method of contacting the test substance with the cells is not particularly limited, and examples thereof include a method of adding the test substance to the medium when using cultured cells. In addition, when the test substance and cells are brought into contact with each other in a non-human animal body, systemic administration such as oral administration, intravenous administration, intramuscular administration, intraperitoneal administration, local administration to target organs and tissues, and intramedullary administration Administration, etc. may be mentioned. In addition, it is preferable to provide a control group that does not come into contact with the test substance.

To measure the expression level of the SPARC gene, the expression level of the SPARC protein may be measured, or the mRNA level of the SPARC gene may be measured.
When measuring the protein expression level, the protein can be extracted from the cell by a known method and quantified by using a known protein expression level measuring method. Examples of the protein expression level measuring method include Western blotting, EIA method, ELISA method, RIA method, and a method using a protein measuring reagent. In addition, the protein expression level may be measured by image analysis of immunostaining as described in Examples described later.
When measuring the amount of mRNA, RNA can be extracted from cells by a known method and quantified using a known method for measuring mRNA amount. Examples of the method for measuring the amount of mRNA include Northern blotting method, RT-PCR method, quantitative RT-PCR method, RNase protection assay and the like.

If the SPARC protein expression level or the SPARC gene mRNA level is increased when the test substance is contacted as compared with the SPARC protein expression level or the SPARC gene mRNA level in the control group not contacted with the test substance, the test concerned. The substance can be selected as the target substance. The extent to which the test substance increases the amount of SPARC protein expression or the amount of SPARC gene mRNA is not particularly limited, but for example, as compared with the amount of SPARC protein expression or the amount of SPARC gene mRNA in cells not in contact with the test substance, for example. It may be 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, and the like.

Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

<Test animal>
ddY mice were obtained from Japan SLC (Shizuoka, Japan).
5XFAD mice, which are transgenic mice, are obtained from The Jackson Laboratory (Bar Harbor, ME, USA), and 5XFAD mice (male, +/-) are wild-type mice (C57BL / 6 * SJL, female, − / −). And genotyping was performed 4-8 weeks after birth. Mice were allowed to freely ingest solid feed and water, and were bred at a constant temperature and humidity (25 ± 2 ° C., 50 ± 5%) and a 12-hour light-dark cycle (light period 7: 00-19: 00).
The 5XFAD mouse is an Alzheimer's model mouse.

<Axotomy>
Craniotomy was performed using a stereotaxic device (manufactured by Narishige (Tokyo, Japan)).
Slowly apply a small blade to the brain of a ddY mouse (female, 8 weeks old) (+ 0.0 mm anterior-posterior (AP), + 1.5 mm media-lateral (ML), -1.6 mm dorsal, as opposed to the previous section. -Ventral (DV)) was invaded and moved in parallel to + 1.5 mm AP. During and after the procedure, the mice were placed on a hot plate to maintain body temperature. The mice were then anesthetized for 1 hour.

<Drug administration>
Diosgenin was obtained from Tokyo Chemical Industry Co., Ltd. Diosgenin was dissolved in the quality standard olive oil (manufactured by Maruishi Pharmaceutical Co., Ltd.) or the solvent (olive oil) of the Japanese Pharmacopoeia.
Diosgenin was orally administered at 0.1 μmol / kg / day to wild-type mice, 5XFAD mice and axotomized ddY mice once daily.

<Retrograde labeling>
0.5 μl of a retrograde tracer, Dextran 3000 MW Texas Red (50 mg / ml in aCSF, Thermo Scientific, Catalog No. D3328) (hereinafter, also simply referred to as “tracer 1”), in front of 5XFAD mice and wild-type mice. It was injected into the prefrontal cortex (+ 1.9 mm AP, + 0.3 mm ML, -2.6 mm DV) at 0.5 ml / min. From 7 days after the injection of Tracer 1, drug administration or AAV9 introduction was performed. Then, 0.5 μl of Dextran 3000 MW FITC (50 mg / ml in aCSF, Thermo Scientific, Catalog No. D3306) (hereinafter, also simply referred to as “tracer 2”) was applied to the prefrontal cortex at the same site as tracer 1 over 7 days. Infused.
In hippocampal CA1 and / or CA3, nerve cells positive only for tracer 1 (red) indicate that axon atrophy has progressed. Nerve cells positive only for Tracer 2 (green) indicate axons extending from the hippocampus toward the prefrontal cortex. Nerve cells positive for both tracers 1 and 2 indicate that the axons did not atrophy or extend.

<Laser capture microdissection (LCM) and DNA microarray>
Mice were deeply anesthetized and immersed in 20 mL of ice-cold saline. The brain was then carefully removed from the skull, snap frozen with dry ice and stored at -30 ° C. The brain was cut to 16 μm in coronal cross section using a cryostat (manufactured by Leica), attached to an RNase-free glass slide, and stored at −80 ° C. until LCM was performed. Using PALM MicroBeam (Carl Zeiss, Oberkochen, Germany), neurons were harvested from one slide within 1 hour at room temperature and axon-stretched from three 5XFAD mice treated with the solvent. Axon-extended neurons 720 were obtained from three 5XFAD mice treated with diosgenin.
RNA was extracted from the collected nerve cells using NucleoSpin RNA (MACHEREYNAGEL GmbH & Co. KG), and T7 RNA polymerase amplification was performed (GeneChip (registered trademark) 3'IVT Pico Kit, ThermoScient). The amplified RNA was hybridized to a mouse Clarium S Array (GeneChip® Hybridization, Wash and Stein Kit, Thermo Scientific). The data was analyzed by the analysis software Transcriptome analog console (Thermo Scientific).

<Western blot>
For mouse hippocampal nerve cells primary cultured on a 35 mm dish, the medium was completely removed 3 days after the start of culture, treated with a solvent or diosgenin, and 4 days later, the cells were rinsed once with PBS and then the protease / phosphatase mixture was used. A lysate was obtained by adding M-PER (Thermo Fisher Scientific) containing a type inhibitor, Protease-Phosphatase inhibitor cocktil (Thermo Fisher Scientific). 5% mercaptoethanol (Wako Pure Chemical Industries, Ltd.) and NuPAGE LDS sample buffer (Thermo Fisher Scientific) were added to lysate (5 μg / lane), and heat treatment was performed at 95 ° C. for 10 minutes to perform SDS-PAGE.
The gel after SDS-PAGE was electrophoresed on a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) for 90 minutes to transfer the protein on the gel to the membrane. Then, the membrane after transfer was immersed in a solution of 5% skim milk (Wako Pure Chemical Industries, Ltd.) and 0.1% Tween 20-Tris Buffered Saline (T-TBS), and the membrane was shaken at room temperature for 1 hour to block. After washing the membrane with T-TBS three times, put Can Get Signal Solution 1 (Toyobo) containing the primary antibody in a rappy bag (Toshin Sangyo) and rotate the bag at 4 ° C overnight with a rotor. It reacted while letting it. After that, the membrane was washed with T-TBS, Can Get Signal Solution 2 (Toyobo) containing a secondary antibody was placed in a rappy bag, and the mixture was reacted in a rotator at room temperature for 2 hours in a light-shielded environment. After that, the membrane was washed with T-TBS, chemiluminescence was detected by LAS4000 (GE healthcare) using ECL Prime Western blotting detection reagent (GE healthcare), and an image was acquired. The brightness of the band was quantified using CS analyzer (ATTO). The primary antibodies used were goat anti-mouse Spark antibody (1: 1000, R & D systems, catalog No. AF942) and mouse anti-GAPDH antibody (1: 1000, Applied biological, secondary antibody, catalog 04). , HRP-conjugated donkey antibody-goat IgG (1: 2000, Santa Cruz) and HRP-conjugated goat antibody-mouse IgG (1: 2000, Santa Cruz).

<Introduction of AAV9 (adeno-associated virus)>
AAV9 vectors were made or purchased from Vector Builder.
AAV9-Syn1-Cerulean-WPRE (Syn1: Synapsin I promoter, Cerulean: blue fluorescence) or AAV9-Syn1-mSpark-IRES-Cerulean-WPRE at 0.5 ml / min, 1 μl / site, 5XFAD mice and wild. -Type mice (female, 7-9 months old) were introduced into Kaiba CA1 (-1.9 mm AP, + 1.7 mm ML, -1.7 mm DV).

<Object recognition memory test>
In this example, the object recognition memory test was performed as follows:
Twenty-one days after the introduction of AAV9, as a training stage, two identical objects that mice saw for the first time were placed in a square box and allowed to perform a 10-minute exploratory action. After a one-hour time interval (interval), a test phase was performed. In the test phase, one of the objects used in the training phase was replaced with a new object (object of different shape and color). At each stage, the number of times the mouse touched the two objects in 10 minutes was recorded. That is, in the test stage, it is a test for confirming whether or not the object seen in the training stage is remembered. In this embodiment, the ratio (%) of the number of searches for a new object to the total search time was calculated as a search-oriented index (Preference index).

<Spatial cognitive memory test>
In this example, the spatial cognitive memory test was performed as follows:
The test was conducted in a relatively lighted room (about 76 lux).
Twenty-three days after the introduction of AAV9, as a training stage, two identical objects that mice saw for the first time were placed in a square box and allowed to perform a 10-minute exploratory action. Of the four walls inside the box, two facing walls will be covered with wallpaper with a characteristic pattern (polka dots and vertical stripes) that will serve as a landmark. After a one-hour time interval (interval), a test phase was performed. In the test phase, one of the objects used in the training phase was placed in a different location from the training phase. At each stage, the number of times the mouse touched the two objects in 10 minutes was recorded. That is, in the test stage, it is a test for confirming whether or not the object seen in the training stage is remembered. In this embodiment, the ratio (%) of the number of searches for a new object to the total search time was calculated as a search-oriented index (Preference index).

In the object cognitive memory test and the spatial cognitive memory test, when the search orientation index exceeds 50%, it indicates that the memory disorder of the mouse is improved.

<Prograde labeling with biotinylated dextran amine (BDA)>
After drug administration to mice, 10% BDA (in PBS, Thermo Scientific, Catalog No. D1956) was applied to hippocampal CA1 (-1.9 mm AP, 6-month-old) in 5XFAD and wild-type mice. +1.7 mm ML, -1.7 mm DV) was injected at a rate of 0.5 ml / min. Seven days later, an immunohistological analysis was performed.

<Immunohistochemical analysis>
Mice were perfused transcardiacly with ice-cooled saline under deep anesthesia, and then the entire brain was removed from the skull and cryopreserved. Brains frozen at −30 ° C. were used in cryostats (CM3050S, Leica, Heidelberg, Germany) to prepare continuous coronal sections with a thickness of 20 μm. After returning the sections to room temperature, the sections were washed with PBS, immersed in a 4% paraformaldehyde (PARAformaldehyde (PFA), Wako Pure Chemical Industries, Ltd.) solution for fixation, a primary antibody solution was added, and the mixture was reacted overnight at 4 ° C. The next day, the sections were washed 3 times with PBS, a secondary antibody solution was added, and the mixture was reacted at room temperature for 2 hours under shading. After washing with PBS three times, it was encapsulated with Aqua Poly Mount. Fluorescence images were acquired using a fluorescence microscope (BZ-X700, KEYENCE). The primary antibodies used were rabbit antibody-NeuN (1: 500, abcam, catalog No. ab104225), goat antibody-mouse SPARC (1: 100, R & D systems, catalog No. AF942), mouse antibody, neurofilament. -H; 1: 250, Coverance, Catalog No. SMI35). Secondary antibodies include Alexa 488-, 568-, 594-, or 647-conjugated affiliate-purified secondary anti-mouse IgG, anti-rabbit IgG, anti-goat IgG, anti-goat IgG, and anti-chicken Ig. 400). In addition, nuclear counterstaining was performed using 4', -6-diamidino-2-phenylindole (DAPI, 1 μg / ml, Sigma-Aldrich).

<Primary nerve cell culture and immunostaining>
For the primary culture of nerve cells, the fetus was removed from a 14-day-old embryonic ddY mouse (Japan SLC, Shizuoka), washed with phosphate buffered saline (PBS), and the medium for primary culture [Neurobasal media]. The hippocampus was excised with 12% horse serum (Life Technologies, 2 mM L-glutamine, 0.6% glucose dissolved) in (Life Technologies, Carlsbad), shredded, suspended, and then seeded as dispersed cultured cells. Culturing was carried out under 10% CO ₂ , 37 ° C. and saturated steam.
For immunostaining of cells, the medium was removed from the treated nerve cells, washed with PBS, and then the nerve cells were fixed with 4% PFA-PBS solution for 90 minutes. The solution was removed and washed twice with 0.3% Triton X-100 (Wako Pure Chemical Industries, Ltd.) -PBS solution for 5 minutes. Primary antibody solution [0.3% TritonX-100-PBS solution, normal got serum (Wako pure drug), mouse anti-pNF-H monoclonal antibody (1: 250, SMI-35, Covance), rabbit anti-MAP2 polyclonal antibody (1) : 2000, Abcam), goat anti-SPARC monoclonal antibody (1: 100, R & D systems)] was added and reacted at 4 ° C. for 24 hours. Remove the solution and remove the secondary antibody solution [0.3% TritonX-100-PBS solution, Alexa Fluor 488-, 568-, 594, 647-labeled goat anti-mouse IgG antibody (1: 300, Molecular Probes, Eugene, OR). , USA) and Alexa Fluor 488-labeled goat anti-rabbit IgG (1: 300, Molecular Probes)] were added and reacted at room temperature for 2 hours under shading. At the same time, nuclear counterstaining was performed using 4', -6-diamidino-2-phenylindole (DAPI, 1 μg / ml, Sigma-Aldrich). After washing with PBS for 5 minutes, the cells were encapsulated with Aqua Poly Mount (Polyscience, Warrington). Fluorescence images were acquired using a fluorescence microscope [BZ-X700 (Keyence) or Cell Observer (Carl Zeiss)]. The length of pNF-H positive axons was automatically traced and the axon length per nerve cell was measured in the region by dividing the axon length by the number of neurons counted by MAP2-positive.

<Image analysis>
Image analysis of axonal extension in the brain of 5XFAD mice revealed the number of tracer-positive, DAPI-positive and NeuN-positive neurons in the hippocampal CA1 and CA3 regions of mice injected with a retrograde tracer in the prefrontal cortex. It was measured using Devices, Sunnyvale). The length of pNF-H-positive axons in primary cultured neurons and the quantification of SPARC proteins expressed in neurons were quantified using MetaMorph (Molecular Devices, Sunnyvale) or Neurocyte (Kurabo). The expression level of SPARC protein limited to axons was quantified using ImageJ. The number of axons positive for anterograde tracers was quantified using ImageJ.

<Statistical analysis>
Statistical ANOVA with GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA), multiple comparison Dunnett's test and Bonferroni test. -Way analyze of variance (ANOVA)], two-way ANOVA with multiple comparison Bonferroni test, and unpaired t-test. It was assumed that p <0.05 was significant. The data are shown as mean ± standard error [standard error of the mean (SEM)].

(Example 1)
As described above, Tracer 1 was injected into the prefrontal cortex of 5XFAD mice (female, 5-6 months old). From 7 days later, the solvent (olive oil) or diosgenin was orally administered for 14 consecutive days. Then, the tracer 2 was injected into the prefrontal cortex at the same site as the tracer 1 as described above. Then, the brain was removed and the number of neurons positive only for Tracer 2 in hippocampus CA1 and CA3 was quantified. The results are shown in FIGS. 1 (CA1) and 2 (CA3) (** p <0.01, *** p <0.001, two-sided test, solvent administration: n number of mice = 3, diosgenin administration: mice. N number = 4).
As shown in FIGS. 1 and 2, in the diosgenin-administered group, there were many neurons that were positive only for tracer 2 (that is, axons extended from the hippocampus to the prefrontal cortex).

From the solvent-administered 5XFAD mouse brain (n = 3) obtained above, LCM was performed as described above, and nerve cells in which axons did not extend were collected.
Further, from the brain (n = 3) of the diosgenin-administered 5XFAD mouse obtained above, LCM was performed as described above, and nerve cells with extended axons were collected.
RNA was extracted from the collected nerve cells as described above, and gene changes in the nerve cells were detected by DNA microarray. Genetic changes in neurons detected by DNA microarrays were compared by Hierarchical scattering and Scatter plot. Since the mRNA expression level of the SPARC gene was the highest in the axon-extended neurons, the gene with the highest increase was identified as the SPARC gene (not shown).

(Test Example 1)
Three days after culturing the hippocampal neurons primary cultured from ddY mice, 1 μM diosgenin or a solvent (0.1% ethanol) was added to the medium, and the cells were cultured in a 35 mm dish for 4 days. Then, lysates were prepared from these nerve cells, and the SPARC protein expression level in each nerve cell was compared with the GAPDH protein expression level in the above-mentioned Western blot. The results are shown in FIG. 3 (** p <0.01, two-sided test, solvent administration: n number of lysates = 7, diosgenin administration: n number of lysates = 7).
As shown in FIG. 3, the SPARC protein expression level was high in the diosgenin-administered group.

(Example 2)
(Expression of SPARC gene)
AAV9-Syn1-Cerulean-WPRE (hereinafter simply referred to as "AAV-Control") at a concentration of 5 × 10 ⁵ , 5 × 10 ⁶ or 5 × 10 ⁷ GC / μl to the hippocampal neurons primary cultured from ddY mice. Or, as a viral vector into which the Spark gene has been introduced, AAV9-Syn1-mSpark-IRES-Cerulean-WPRE (hereinafter, also simply referred to as “AAV-Spark”) was treated for 7 days.
The expression level of SPARC protein in MAP2-positive neurons was quantified by the above immunostaining and image analysis. The results are shown in FIG. In FIG. 4, noAAV indicates a group in which no treatment was performed (***** p <0.0001 vs. AAV-Control, one-way ANOVA post hoc Bonferroni test, n of nerve cells. Number = 337-558).
As shown in FIG. 4, it was confirmed that the SPARC gene was upregulated in the cells treated with AAV-Spark.

(Axon extension)
Further, for the cells treated with AAV-Control, AAV-Parc (5 × 10 ⁶ GC / μl), or nothing, the images of nerve cells were obtained by the above image analysis (FIG. 5), and pNF. -The length of H-positive axons was quantified. The results are shown in FIG. 6 (* p <0.05, one-way ANOVA post hoc Bonferroni test, n number of images = 10 to 16).
As shown in FIG. 6, axons were most extended in mice treated with AAV-SPARC.

(Example 3)
AAV-Control or AAV-Parc 10 ¹⁰ GC was injected into hippocampal CA1 of wild-type and 5XFAD mice (female, 7-9 months old). An object cognitive memory test was performed 21 days after the injection, and a spatial cognitive memory test was performed 23 days after the injection. The results are shown in FIGS. 7 and 8 (** p <0.01, *** p <0.001, one-way ANOVA post hoc Dunnett's test. Gene treatment and memory test by repeated two-way ANOVA. It was shown that there was a significant difference in the interaction between [F (2,23) = 13.77, p = 0.0018 (FIG. 7), [F (2,23) = 16.33, p = 0.0010 (FIG. 8). #### p <0.001, #### p <0.0001, post hoc Bonferroni test, n number of mice = 4). In FIGS. 7 and 8, Cont indicates an AAV-Control treatment group, and Spark indicates an AAV-Parc treatment group.
As shown in FIGS. 7 and 8, it was confirmed that the memory impairment was improved by the AAV-SPARC treatment.

After the memory test was completed, the mouse brain was excised and a brain section was prepared. In hippocampal CA1, the amount of infused AAV9-derived Cerulean (blue) fluorescence and Spark protein (red) in NeuN-positive neurons (green) was detected by fluorescent immunostaining. It was confirmed that the AAV-SPARC treatment detected more red color than the AAV-Control treatment, and that the expression of the SPARC gene was more induced (not shown).

(Example 4)
As described above, Tracer 1 was injected into the prefrontal cortex of wild-type and 5XFAD mice (female, 7-9 months old). Seven days later, AAV-Control or AAV-Parc 10 ¹⁰ GC was applied to hippocampal CA1 for 21 consecutive days. Then, the tracer 2 was injected into the prefrontal cortex at the same site as the tracer 1 for 7 days, and then the brain was removed. In the hippocampal CA1 of the excised brain, the number of neurons positive only for tracer 2, the number of neurons positive only for tracer 1, and the number of neurons positive for both tracers 1 and 2 were quantified. The results are shown in FIGS. 9 to 11 (* p <0.05, *** p <0.001, *** p <0.0001, one-way ANOVA post hoc Bonferroni test, n number of mice = 4). ). In FIGS. 9 to 11, Cont indicates an AAV-Control treatment group, and Spark indicates an AAV-Parc treatment group.
As is clear from FIGS. 9 to 11, in the AAV-Parc treatment group, there were many neurons that were positive only for Tracer 2 (that is, axons extended from the hippocampus to the prefrontal cortex).

(Example 5)
The solvent (olive oil) or diosgenin was orally administered to wild-type mice and 5XFAD mice at 0.1 μmol / kg / day for 21 consecutive days. On day 14 of drug administration, the anterograde tracer BDA was injected into hippocampal CA1. Seven days after BDA injection, mouse brain sections were prepared and fluorescent immunostaining detected BDA-positive axons (red), SPARC proteins (green), and DAPI (blue) in the prefrontal cortex. Since the BDA antegradely extends to the prefrontal cortex, axons projected from the hippocampus toward the prefrontal cortex are labeled with the BDA. The results of quantifying the number of BDA-positive axons are shown in FIG. 12 (* p <0.05, *** p <0.001, one-way ANOVA post hoc Bonferroni test, for the solvent-administered group of 5XFAD mice). N number of mice = 5). In FIG. 12, Veh indicates a solvent-administered group, and Dios indicates a diosgenin-administered group.
As shown in FIG. 12, in the diosgenin-administered group, there were many nerve cells in which axons extended from the hippocampus to the prefrontal cortex.

(Test Example 2)
Primary cultured neurons isolated from ddY mice (14th day of embryonic development) are cultured for 3 days and treated with Aβ25-35 (amyloid β protein fragment 25-35) (2.5 μM), which is the causative agent of Alzheimer's disease, for 3 days. did. Then, the medium containing Aβ25-35 was removed, treated with a solvent or diosgenin (0.1 μM or 1 μM) for 4 days, and subjected to fluorescent immunostaining. Image analysis confirmed that the SPARC protein expression level was increased especially on the axons and at the axon terminals in the diosgenin-administered group (not shown). In addition, the SPARC protein expression level per axon was quantified by the above-mentioned immunostaining and image analysis (* p <0.05, *** p <0.001, for Aβ / Veh group, multiple comparison Dunnett's test). One-way ANOVA post hoc Dunnett's tes by test, n number of axons = 92-427). The results are shown in FIG.
In the axons of nerve cells treated with Aβ25-35, the expression level of SPARC protein was increased in the diosgenin-administered group.
In addition, in FIG. 13, since the expression level of the SPARC protein in the axon is lower in the Aβ / Veh group than in the control group (Cont), the SPARC protein is decreased in the atrophied axon. I understand.

(Example 6)
Tracer 1 (Dextran Texas Red) was injected into the prefrontal cortex of Wild-type and 5XFAD mice, and 7 days later, AAV-Control or AAV-Spark was injected into 10 ¹⁰ GC and hippocampal CA1 respectively. Twenty-one days after AAV injection, Tracer 2 (Dextran FITC) was injected into the prefrontal cortex, and the brain was removed 7 days later.
Quantitative values of the number of neurons (the number of Nexus Red-positive neurons) that were axillarily projected from the hippocampus CA1 to the frontal prefield before AAV injection are shown in FIG. 14A. Quantitative values of the number of nerve cells that were searched and projected (the number of FITC-positive nerve cells) are shown in FIG. 14B (***** p <0.001, *** p <0.0001, one-way ANOVA post hoc). Bonferroni test, n number of mice = 4).
FIG. 14A shows the number of hippocampal CA1 neurons extending axons to the prefrontal cortex prior to overexpression of the SPARC gene. Compared to wild-type mice, the two groups of 5XFAD mice are equally less. That is, before the expression of the SPARC gene, about 40% of the axons were similarly atrophied. FIG. 14B shows a total of 28 days after the SPARC gene was overexpressed. In mice expressing only the 5XFAD control vector, the number of cells extending axons to the prefrontal area remains smaller than in wild-type mice, but in the SPARC gene expression group, the number of nerve cells projecting exceeds the wild type. Increased. This result indicates that all of the atrophied axons in hippocampal CA1 extended toward the prefrontal cortex.

(Example 7)
Tracer 1 (Dextran Texas Red) was injected into the prefrontal cortex of Wild-type and 5XFAD mice, and 7 days later, AAV-Control or AAV-Spark was injected into 10 ¹⁰ GC and hippocampal CA3, respectively. Twenty-one days after AAV injection, Tracer 2 (Dextran FITC) was injected into the prefrontal cortex, and the brain was removed 7 days later.
Quantitative values of the number of neurons (the number of Nexus Red-positive neurons) that were axillarily projected from the hippocampus CA3 to the frontal prefield before AAV injection are shown in FIG. 15A. Quantitative values of the number of neurons projecting (FITC-positive neurons) are shown in FIG. 15B (** p <0.01, *** p <0.001, one-way ANOVA post hoc Bonferroni test). , N number of mice = 4).

FIG. 15A shows the number of hippocampal CA3 neurons extending axons to the prefrontal cortex prior to overexpression of the SPARC gene. Compared to wild-type mice, the two groups of 5XFAD mice are equally less. That is, before the expression of the SPARC gene, about 40% of the axons were similarly atrophied. FIG. 15B shows a total of 28 days after the SPARC gene was overexpressed. In mice expressing only the 5XFAD control vector, the number of cells extending axons to the prefrontal area remains smaller than in wild-type mice, but in the SPARC gene expression group, the number of nerve cells projecting exceeds the wild type. Increased. This result indicates that all of the atrophied axons in hippocampal CA3 extended toward the prefrontal cortex.

The agents or compositions of the present invention are extremely useful because they can treat or ameliorate diseases associated with axonal dysfunction of nerve cells.

Claims (10)

An agent or composition for axonal extension of nerve cells, which comprises a component (A) that expresses or promotes the expression of SPARC protein (however, component (A) excludes diosgenins). An agent or composition for extending axons of nerve cells to a projection destination region, which comprises a component (A) that expresses or promotes the expression of SPARC proteins. The agent or composition according to claim 2, wherein the axon is the axon of a hippocampal nerve cell, and the projection destination region is the prefrontal cortex. An agent or composition for use in the following uses (1) and / or (2), which comprises a component (A) that expresses or promotes the expression of a SPARC protein (provided that the component (A) is , Excluding diosgenins).
(1) Treatment and / or improvement of diseases associated with neuronal axonal dysfunction (2) Improvement and / or maintenance of memory The agent or composition according to claim 4, wherein the disease associated with the dysfunction of the axon of a nerve cell is one or more selected from Alzheimer's disease, spinal cord injury, cerebral contusion, Parkinson's disease and dementia. The agent or composition according to any one of claims 1 to 5, wherein the component (A) contains one or more components selected from the following (A-1) to (A-4).
(A-1) Gene encoding SPARC protein (A-2) One or more enzymes selected from PI3 kinase, MEK1, protein kinase A and protein kinase C (A-3) The enzyme of (A-2) above. Proteins or peptides that activate one or more in nerve cells, vitamins that activate one or more of the above (A-2) enzymes in nerve cells, and one or more of the above (A-2) enzymes in nerve cells. Transcription of one or more enzyme-activating components (A-4) selected from growth factors or growth factors that are activated in the above and steroids that activate one or more of the above-mentioned enzymes (A-2) in nerve cells. One or more transcription-activating components selected from a component that activates transcription via the factor c-Jun and a component that activates transcription via the transcription factor c-Jun and the transcription factor Fra1. The agent or composition according to any one of claims 2 to 3 and 6, wherein the component (A) contains diosgenins. A method for screening an axonal extender for nerve cells, which comprises selecting a component that promotes the expression of a SPARC protein. A method of screening an agent for extending axons of nerve cells to a projection destination region, which comprises selecting a component that promotes expression of a SPARC protein. A method for screening an agent for use in the following uses (1) and / or (2), which comprises selecting a component that promotes the expression of a SPARC protein.
(1) Treatment and / or improvement of diseases associated with neuronal axonal dysfunction (2) Improvement and / or maintenance of memory

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