Screening methods for dementia medications to improve memory

Abstract

To provide a method for screening a medicine to treat dementia by improving memory.SOLUTION: The invention is based on the finding that Nedd4-2, which is one of E3 (ubiquitin ligase) that determines substrate specificity of ubiquitination, suppresses memory capacity. A postsynaptic transmembrane protein, Prr7, is identified as a substrate for Nedd4-2. As a result, it is found that a candidate therapeutic agent for dementia is obtained by screening a substance that inhibit ubiquitination of Prr7 by Nedd4-2 to complete the invention.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for screening drugs for treating dementia to improve memory.

Background Art

[0002] In the research and development of drugs for treating dementia typified by Alzheimer's disease and vascular dementia, a strategy has been taken to develop a radical treatment method after elucidating the cause of the disease. For example, for Alzheimer's disease, treatments using antibodies against amyloid-β, which is considered to be the cause, have been studied. However, with this strategy, no significant achievements in the treatment of dementia have been obtained so far. In addition, drugs for treating dementia targeting proteins other than amyloid-β have not been developed so far.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] Since the development of a radical treatment method for dementia has not progressed, the development of a symptomatic treatment method for memory impairment has become important. The present invention, unlike the conventional dementia treatment strategies, aims to provide a method for screening a drug that can treat dementia by improving a specific symptom, that is, by improving memory.

Means for Solving the Problems

[0005] Memory is controlled at synapses within nerve cells. To address the above problem, the inventors have diligently studied the mechanisms of synapse formation and function, particularly the ubiquitination-mediated control mechanism. They discovered that Nedd4-2, one of the E3 (ubiquitin ligases) that determine the substrate specificity of ubiquitination, suppresses memory capacity. Furthermore, they identified Prr7, a postsynaptic transmembrane protein, as a substrate of Nedd4-2. As a result, the inventors believe that by screening for substances that inhibit Prr7 ubiquitination by Nedd4-2, they can develop therapeutic drugs for dementia. We found that we could obtain candidate substances. Furthermore, by deleting Nedd4-2, We completed the present invention by discovering that long-term potentiation of synaptic transmission increases and / or spatial learning ability is enhanced.

[0006] In other words, the present invention is as follows: [1] A method for screening for therapies for dementia, characterized in that candidate therapeutic substances are selected using the ability to inhibit the ubiquitination of Prr7 by ubiquitin-modifying enzymes as an indicator. [2] The method according to [1], wherein the ubiquitin-modifying enzymes include E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and Nedd4-2. [3] The method according to [2], comprising the following steps. (A) A step of contacting the Prr7 protein immobilized on the surface of a substrate with a solution containing labeled ubiquitin, E1, E2 and Nedd4-2. (B) A step of adding a test substance to the solution that has been brought into contact with the immobilized Prr7 protein. (C) Further, ATP is added to the solution and incubated under conditions that promote the ubiquitination reaction. The process of baiting, and (D) A step of selecting a candidate therapeutic agent based on the indicator that the label is not detected, or that the detected value of the label is lower than the measured value in the negative control substance or a predetermined cutoff value. [4] The method according to [3], further comprising the step of immobilizing (A')Prr7 protein on the surface of a substrate. [5] The method according to [3] or [4], wherein the substrate is a multiwell plate. [6] The method according to any one of [3] to [5], wherein the label is a fluorescent label. [7] Deficiency of Nedd4-2 increased long-term potentiation of synaptic transmission and / or Genetically modified animals (excluding humans) with enhanced spatial learning abilities. [Effects of the Invention]

[0007] This invention provides a method for screening for pharmaceuticals that can treat dementia by improving memory. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows the strategy for creating Nedd4-2 deficient mice. [Figure 2] This shows the localization of Nedd4-2 within spines. A is a photograph of primary cultured hippocampal neurons labeled with Nedd4-2 (upper panel) or GFP (lower panel) and observed using stimulated release inhibition (STED) microscopy. B is a schematic diagram showing the ubiquitination of Prr7 by Nedd4-2 in the postsynaptic region. [Modes for carrying out the invention]

[0009] The embodiments will be described in detail below with reference to the attached drawings, but this does not limit the scope of the invention as claimed.

[0010] <Screening Method> One embodiment of the present invention is a screening method for dementia treatment drugs, characterized by selecting candidate therapeutic substances using the ability to inhibit the ubiquitination of Prr7 by ubiquitin-modifying enzymes as an indicator.

[0011] Prr7 is a transmembrane protein at the postsynaptic part, and its specific sequence is not particularly limited. For example, proteins having the amino acid sequence of SEQ ID NO: 1 or 2 can be mentioned. Also, as long as Prr7 has the property as a gene related to intelligence, it may be a protein in which one or more amino acid sequences are deleted, substituted , added, or inserted. Here, one or more means, for example, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 to 2, etc. Also, Prr7, for example, may be a protein having an amino acid sequence that is 90% or more, preferably 95% or more, more preferably 98% or more identical to the amino acid sequence represented by SEQ ID NO: 1 or 2 and having the property as a gene related to intelligence.

[0012] The ubiquitin-modifying enzyme is not particularly limited as long as it can ubiquitinate Prr7, but it is preferably one that includes E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and Nedd4-2. Specific sequences are not particularly limited. For example, for E1, a protein having the amino acid sequence of SEQ ID NO: 3 can be mentioned, for E2, a protein having the amino acid sequence of SEQ ID NO: 4 can be mentioned, and for Nedd4-2, a protein having the amino acid sequence of SEQ ID NO: 5 can be mentioned. Also, as long as E1, E2, and Nedd4-2 can ubiquitinate Prr7 in combination, they may be proteins in which one or more amino acid sequences are deleted, substituted, added, or inserted. Here, one or more means, for example, in the case of E1, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 to 2, etc. can be mentioned, in the case of E2, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 to 2, etc. can be mentioned, and in the case of Nedd4-2, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5, 1 to 3, or 1 to 2, etc. can be mentioned. Examples include ~2. In addition, E1, E2, and Nedd4-2 have an amino acid sequence identical to 90% or more, preferably 95% or more, more preferably 98% or more of the amino acid sequences represented by SEQ ID NOs: 3 to 5, respectively, and by using E1, E2, and Nedd4-2 in combination, ubiquitination of Prr7 can be performed. It may also be a protein capable of performing ubiquitination.

[0013] The screening method of this embodiment can include the following steps (A) to (D). (A) A step of contacting a solution containing a Prr7 protein immobilized on the surface of a substrate with a labeled ubiquitin, E1, E2, and Nedd4-2. (B) A step of adding a test substance to the solution that has been contacted with the immobilized Prr7 protein. (C) Further adding ATP to the solution and incubating under conditions that promote the ubiquitination reaction. Step, and (D) A step of selecting a therapeutic agent candidate substance using as an index that the label is not detected, or that the detected value of the label is lower compared to the measured value in a negative control substance or a predetermined cut-off value.

[0014] Furthermore, it may include (A’) a step of immobilizing the Prr7 protein on the surface of the substrate. Step (A’) is usually performed before step (A).

[0015] Also, as long as the screening of a therapeutic agent for dementia that inhibits the ubiquitination of Prr7 is not hindered, it may further optionally include additional steps. For example, after immobilizing the Prr7 protein on the substrate, a washing step can be optionally added.

[0016] In step (A), the substrate is not particularly limited as long as the screening of a therapeutic agent for dementia that inhibits the ubiquitination of Prr7 is not hindered. For example, a 96-well plate or a 384-well plate. While multi-well plates such as tents, petri dishes, and beads can be used, multi-well plates are preferred from the viewpoint of being able to process a large number of samples simultaneously. Furthermore, the material of the base material is not particularly limited, and examples include polyethylene, polystyrene, and polypropylene.

[0017] The method for immobilizing the Prr7 protein onto the substrate is not particularly limited and can be carried out by methods known to those skilled in the art. Alternatively, a commercially available substrate with the Prr7 protein already immobilized on it may be used.

[0018] The amount of Prr7 protein immobilized on the substrate is not particularly limited as long as it does not inhibit the ubiquitination reaction, and is not particularly limited as long as it is not excessively large or small compared to the amounts of ubiquitin E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), Nedd4-2, and the test substance, etc. stomach.

[0019] After the Prr7 protein has been immobilized onto the substrate, it may be appropriately blocked with skim milk, albumin, casein, etc., by methods known to those skilled in the art, in order to prevent nonspecific adsorption.

[0020] The labeling is not particularly limited as long as it does not interfere with the screening of dementia treatments that inhibit the ubiquitination of Prr7, and fluorescent labels such as FITC can be used, for example.

[0021] The amount of labeled ubiquitin, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and Nedd4-2 added is not particularly limited as long as it does not inhibit the ubiquitination reaction, and is not fixed to the substrate. The amount of phased Prr7 protein is not particularly limited, as long as it is not excessively large or small relative to the test substance.

[0022] Regarding the solution, the type of solvent is not particularly limited as long as it does not inhibit the ubiquitination reaction; for example, water or any buffer solution can be used. The pH of the solution may be adjusted as appropriate.

[0023] The preparation of a solution containing labeled ubiquitin, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and Nedd4-2 may be done in advance, or, for example, the Prr7 protein may be used. After adding a solvent to a substrate such as a multi-plate with a solidified surface, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and Nedd4-2 are added to the solvent on the substrate. You can go that way.

[0024] The contact time and temperature are not particularly limited as long as they do not inhibit the ubiquitination reaction, but are preferably 20 to 40°C, and more preferably 30 to 37°C.

[0025] In step (B), the test substance may be a low-molecular-weight compound, an antibody, etc. When the test substance is a low-molecular-weight compound, it is not particularly limited, but for example, low-molecular-weight compounds obtained from the low-molecular-weight compound library of the RIKEN Natural Compound Bank (NPDepo) can be used. Furthermore, when the test substance is an antibody, although not particularly limited, for example, the Nedd4-2 binding site of Prr7. A recombinant antibody that recognizes the region (SEQ ID NO: 6) can be used, and it is preferable that this recombinant antibody is a nanobody antibody.

[0026] The amount of test substance added is not particularly limited as long as it does not inhibit the ubiquitination reaction, nor is it particularly limited as long as it is not excessively large or small relative to the amount of Prr7 protein immobilized on the substrate or the test substance.

[0027] Steps (A) and (B) may be performed sequentially, or steps (A) and (B) may be performed simultaneously. In addition, step (A) may be performed after step (B), that is, the Prr7 protein immobilized on the surface of the substrate may be brought into contact with a solution containing the test substance, and then the solution that was in contact with the immobilized Prr7 protein may be brought into contact with a solution containing labeled ubiquitin, E1, E2, and Nedd4-2. When steps (A) and (B) are performed simultaneously, the preparation of the solution containing labeled ubiquitin, E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), Nedd4-2, and the test substance is as follows: This may be done in advance, or it may be done by adding a solvent to a substrate such as a multiplate on which the Prr7 protein is immobilized on the surface, and then adding E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), Nedd4-2 and the test substance to the solvent on the substrate.

[0028] In step (C), the amount of ATP added is not particularly limited as long as it does not inhibit the ubiquitination reaction. Furthermore, it is not particularly limited as long as the amount of Prr7 protein immobilized on the substrate is not excessively large or small relative to the test substance.

[0029] Incubation is not limited to specific temperatures or times, as long as it is carried out under conditions that promote the ubiquitination reaction; for example, it can be done at 37°C for 20 minutes to 1 hour.

[0030] In step (D), the negative control substance is a substance that does not inhibit the ubiquitination reaction. For example, a substance added to the well in which a label was detected in the same multi-well plate may be used as the negative control substance, or a substance that has been shown in advance not to inhibit the ubiquitination reaction may be used as the negative control substance. Alternatively, a substance that inhibits ubiquitination may be used as the positive control substance. For example, a known low-molecular-weight compound or antibody that inhibits ubiquitination may be used. The detected label is a ubiquitin label bound to the Prr7 protein immobilized on the substrate surface, and is usually washed after incubation in step (C).

[0031] In this embodiment, the dementia treatment drug improves memory, and indicators known to those skilled in the art can be used to determine the improvement in memory. For example, it can be determined by an increase in long-term potentiation of synaptic transmission or an enhancement of spatial learning ability.

[0032] The screening method of this embodiment is high-throughput, and it is possible to simply and efficiently find a candidate therapeutic substance for the purpose, and such a candidate therapeutic substance can be applied to the use as a medicine for treating dementia.

[0033] Regarding the increase in long-term potentiation of synaptic transmission and the enhancement of spatial learning ability, etc., the effects can be confirmed in an animal model.

[0034] <Nedd4-2 Deficient Animals and Method for Producing the Same> Another embodiment of the present invention is a genetically modified animal and a method for producing the same, in which long-term potentiation of synaptic transmission is increased and / or spatial learning ability is enhanced by deleting Nedd4-2.

[0035] The Nedd4-2 deficient mouse (Nedd4-2 knockout mouse) of this embodiment is a mouse in which the function of the Nedd4-2 protein is deficient because the Nedd4-2 gene on the chromosome is replaced by an inactive Nedd4-2 gene. The "inactive Nedd4-2 gene" refers to a gene that cannot express a normal Nedd4-2 protein due to a partial deletion of the Nedd4-2 gene, insertion of another base sequence into the coding region of the Nedd4-2 gene, etc. Examples of the deficient Nedd4-2 gene include a gene in which exon 15 is deleted etc., but are not limited thereto. "The function of the Nedd4-2 protein is deficient <l " means that the function of the Nedd4-2 protein as a ubiquitin ligase is lost, preferably, it means that the function of the Nedd4-2 protein is completely lost, but it also includes cases where only one allele is replaced by an inactive form as in a heterozygous knockout mouse and the function of the Nedd4-2 protein is partially lost.

[0036] ​An example of the Nedd4-2 gene is the mouse Nedd4-2 gene with the nucleotide sequence shown in Sequence ID No. 7. This nucleotide sequence is registered in GenBank with accession number NM_001114386.1, and information on the coding region and exons can be found by referring to this number. It can be obtained by [method]. Note that the Nedd4-2 gene differs depending on the species, so the sequence number It may also be a homolog gene of gene 7. The homolog gene is located on the mouse chromosome Nedd4-2 Examples of homologous genes include those with a degree of homology that allows for homologous recombination with the gene, for example, those having 80% or more, preferably 90% or more, and more preferably 95% or more homology with the base sequence of Sequence ID No. 7. Furthermore, the homologous gene may be a gene that hybridizes with the gene having the base sequence of Sequence ID No. 7 under stringent conditions. Here, stringent conditions include, for example, performing hybridization and washing at 65°C, 1×SSC, 0.1% SDS, preferably 65°C, 0.1×SSC, 0.1% SDS.

[0037] The Nedd4-2 gene-deficient mouse of the present invention can be modified using known genetic engineering methods (gene targeting). It can be manufactured by the (synthesis) method. For example, it can be manufactured as follows. First, a partial fragment of the Nedd4-2 gene is prepared, and a targeting vector is created to replace the Nedd4-2 gene with a deletion type. For the selection of recombinants, it is preferable to incorporate drug resistance genes into the targeting vector. Neomycin resistance genes, hygromycin B phosphotransferase genes, etc., can be used as marker genes for drug selection. Additionally, to delete a partial sequence of the Nedd4-2 gene, there are systems such as the Cre-loxP system (R. Kuhn et al. Science, 269, 1427-1429, 1995) and the FLP / FRT system (Rodriguez et al. Nat Gene). (t 25:139-40.) may also be used. In that case, the subsequence to be deleted is the loxP sequence or FRT The targeting vector is constructed so that it is sandwiched between the array elements.

[0038] Below, we obtain Nedd4-2 gene-deficient mice using the targeting vector described above. A general method for obtaining the present invention is described below. However, the mice of the present invention are not limited to those obtained by the following methods. Homologous recombination is performed using the targeting vector prepared by the method described above. Embryonic stem cells (ES cells) can be used for homologous recombination. Several mouse-derived ES cell lines have been established, and CCE cell line, TT2 cell line, AB-1 cell line, J1 cell line, R1 cell line, E14TG2a cell line, etc. can be used. The targeting vector is prepared by known methods. It can then be introduced into mouse ES cells. For example, electroporation, liposomes, calcium phosphate, and DEAE-dextran methods can be used. Next, cell clones in which the wild-type Nedd4-2 gene on the chromosome is replaced with the mutant Nedd4-2 gene in the targeting vector are selected. Selection can be based on drug resistance, etc., and it is preferable to further confirm homologous recombination by Southern blotting or PCR. It's nice.

[0039] The ES cells containing the mutated gene obtained in this way are introduced into the embryos of wild-type mice. Then, these ES cell-introduced embryos are transplanted into the uterus of a surrogate mother mouse in a pseudopregnancy state, and by allowing the mouse to give birth, a chimeric animal can be created. Microinjection and agglutination methods are known for introducing ES cells into embryos such as blastocyst-stage embryos, but microinjection is preferred. Pseudopregnancy female mice to be used as surrogate mothers can be obtained by mating a female mouse with a normal cycle with a male mouse that has been castrated by methods such as vas deferens ligation.

[0040] Next, these chimeric mice are crossbred with purebred mice to create mice derived from ES cells through the germline. Animals in which the recombinant ES cells transplanted into the embryo have transitioned to the germline are selected, and these animals are bred to obtain heterozygotes lacking the Nedd4-2 gene. It is possible. By mating the obtained Nedd4-2 gene-deficient heterozygous mice with each other, Nedd4-2 gene-deficient homozygous mice can be obtained.

[0041] Furthermore, in preparing the knockout mice of the present invention, the Cre / loxP system and FLP / FRT system, which are commonly used in the preparation of conditional knockout mice, are not used. It is also possible to use a TEM. When using the Cre / loxP system, the Cre recombinant enzyme is sandwiched between loxP sequences by mating with an animal expressing Cre recombinant enzyme derived from E. coli P1 phage, or by infecting with a viral vector containing the Cre gene. Because it recognizes and removes the rare sequence, it is possible to create Nedd4-2 gene-deficient mice. Furthermore, by crossing mice that express tissue-specific Cre recombinant enzymes with mice that express them, It is also possible to create knockouts with the characteristic of a specific gene deficiency. Similarly, when using the FLP / FRT system, Nedd4-2 gene-deficient mice can be produced by crossing them with animals that express FLP recombinant enzyme.

[0042] Nedd4-2 gene-deficient mice, as described above, exhibit increased long-term potentiation of synaptic transmission and / or enhanced spatial learning ability compared to mice that do not lack the Nedd4-2 gene. Therefore, these mice can be used to elucidate the mechanisms of dementia onset and to develop treatments.

[0043] The animal is not particularly limited to any mammal, but it is preferable that it be a mammal other than a human. [Examples]

[0044] The present invention will be described below using examples, but the present invention is not limited to these examples.

[0045] [Example 1: Creation of Nedd4-2 deficient mice] The Nedd4-2 Floxed mouse was prepared using a recombineering system known to those skilled in the art (non- Patent Document 1). The strategy for generating Nedd4-2-deficient mice is shown in Figure 1. First, a mouse genome containing exons 14 to 17 of Nedd4-2 was cloned into the pL253 vector. Subsequently, the LoxP sequence was inserted to flank the 15th exon. This vector was introduced into mouse ES cells, and only ES cells that underwent homologous recombination were cloned. Mice were bred from these ES cell clones and crossed with mice expressing FLIP recombinase to eliminate the Neo cassette from the genome. The resulting Nedd4-2 Floxed mice and NEX-Cre mice were then crossed. Neuron cell-specific Nedd4-2 deficient mice were created by crossing them with Us (Non-Patent Literature 2).

[0046] [Example 2: Ubiquitination and fractionation of Prr7 by Nedd4-2 in postsynaptic spines] [Involvement in the solution] Primary hippocampal cultured cells from wild-type mice were observed using a STED microscope, with GFP and Nedd4-2 as indicators. The results showed that Nedd4-2 accumulated in postsynaptic spines labeled with GFP (indicated by arrowheads in Figure 2A). Furthermore, hippocampal slices with a thickness of 300 μm were prepared from neuronal cell-specific Nedd4-2 knockout mice (Nedd4-2 nKO), and extracellular local field potentials were measured using methods known to those skilled in the art. As a result, it was revealed that long-term potentiation of synaptic transmission was increased in the hippocampal CA1 region. Behavioral experiments using the Morris water maze test with mice aged 8 weeks to 5 months showed that spatial learning ability was enhanced. Nedd4-2 is a ubiquitous expression in the central nervous system. It is a ubiquitinating enzyme, and increased ubiquitination by Nedd4-2 is associated with a decline in memory and learning ability. It is thought that Nedd4-2 is involved in the increase of long-term potentiation, and as a substrate of Nedd4-2, postsynaptic We identified the transmembrane protein Prr7. Human Prr7 has previously been reported as a gene associated with intelligence (Non-Patent Literature 3). Prr7 protein expression levels were increased in Nedd4-2 nKO. This is thought to be because the absence of Nedd4-2 prevents Prr7 from being ubiquitinated and therefore not degraded. flox We created Nedd4-2 nKO;Prr7 heterozygote mice by crossing Nedd4-2 nKO mice with other Nedd4-2 nKO mice. These mice lacked Nedd4-2, but Prr7 expression levels were reduced to nearly the same level as wild-type mice. Analysis of these mice revealed that long-term potentiation of synaptic transmission was nearly the same as in wild-type mice. Therefore, the increased Prr7 expression in Nedd4-2 nKO mice is essential for enhancing synaptic plasticity. This suggests that the ubiquitination and degradation of Prr7 inhibit synaptic plasticity and memory. Based on these results, it is thought that inhibiting Prr7 ubiquitination can restore memory in dementia patients. These results indicate that Nedd4-2 ubiquitinates Prr7 in postsynaptic spines. This suggests that it leads to decomposition, which in turn suggests that long-term potentiation of synaptic transmission and spatial learning ability are suppressed (Figure 2B).

[0047] [Example 3: Screening method for developing dementia treatments that pharmacologically inhibit the ubiquitination of Prr7] The specific screening procedure for small molecules that inhibit Prr7 ubiquitination is as follows: Prr7, a substrate for ubiquitination, is adsorbed to the bottom of the wells of a 96-well plate. To monitor the ubiquitination reaction of Prr7, FITC-labeled ubiquitin (FITC-ubiquitin) is used in combination. The purified replacement protein (Nedd4-2, which consists of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin ligase)) was used in a 96-well plate adsorbed with Prr7. It is added to the solution. Additionally, small molecule compounds that inhibit the ubiquitination of Prr7 are screened. To achieve this, a library of small molecule compounds was obtained from the RIKEN Natural Compound Bank (NPDepo), and each individual small molecule compound contained in that library was added to each well of the 96-well plate. Subsequently, ATP was added to each well, and the ubiquitination reaction was carried out at 37°C. After the ubiquitination reaction is complete, the fluorescence intensity of FITC at the bottom of each well is measured. When the fluorescence intensity of FITC decreases compared to wells where Prr7 has been ubiquitinated, the small molecule compound added to the well with decreased FITC fluorescence intensity is identified as a promising candidate for inhibiting Prr7 ubiquitination, and furthermore, as a promising candidate for treating dementia.

[0048] Furthermore, the small molecule compounds obtained through the above screening method can be used in the development of groundbreaking dementia treatments that aim to restore memory by inhibiting the specific ubiquitination reaction of Prr7.

Claims

1. A method for screening for therapies for dementia, characterized in that candidate therapeutic substances are selected using the ability to inhibit the ubiquitination of Prr7 by a ubiquitin-modifying enzyme as an indicator, wherein the ubiquitin-modifying enzyme includes Nedd4-2.

2. The method according to claim 1, wherein the ubiquitin-modifying enzyme further comprises E1 (ubiquitin-activating enzyme) and E2 (ubiquitin-conjugating enzyme).

3. The method according to claim 2, comprising the following steps. (A) A step of contacting the Prr7 protein immobilized on the surface of a substrate with a solution containing labeled ubiquitin, E1, E2, and Nedd4-2. (B) A step of adding a test substance to the solution that has been brought into contact with the immobilized Prr7 protein. (C) Further, ATP is added to the solution and incubated under conditions that promote the ubiquitination reaction. The process of tying, (D) A step of selecting a candidate therapeutic agent based on the fact that the label is not detected, or that the detected value of the label is lower than the measured value in the negative control substance or a predetermined cutoff value.

4. (A') The method according to claim 3, further comprising the step of immobilizing the Prr7 protein on the surface of a substrate.

5. The method according to claim 3 or 4, wherein the substrate is a multiwell plate.

6. The method according to any one of claims 3 to 5, wherein the label is a fluorescent label.

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