Expression construct for protein for enhancing spines, transformed cells, transgenic non-human animals, drugs for treating or preventing mental disorders, cognitive dysfunction, sleep disorders, or memory disorders, and methods for enhancing spines

Abstract

To provide an expression construct for a protein for enhancing spines that can be introduced into neurons to increase a spine volume.SOLUTION: Provided are: [1] an expression construct for a protein for enhancing spines including a promoter for expression that functions in nerve cells, a domain of a Kalirin-7 protein required for at least spine enhancement, operably linked to the promoter, and a nucleotide sequence encoding a protein J1 that forms a heterodimer in the presence of a dimer-inducing compound; and [2] an expression construct for a protein for enhancing spines including a promoter for expression that functions in nerve cells, a domain of a PSD-95 protein required for the localization to at least a postsynaptic membrane, operably linked to the promoter, and a nucleotide sequence encoding a protein J2 that forms a heterodimer in the presence of a dimer-inducing compound.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an expression construct for a protein for enhancing spines, a transformed cell, a transgenic non-human animal, a drug for treating or preventing a mental disorder, and a method for enhancing spines, which utilize the spine-enhancing effect of Kalirin-7 protein. [Background technology]

[0002] In the field of neuroscience, detailed analyses of neurons are being carried out to understand the activities within the brain and to elucidate the mechanisms that maintain normal brain development and proper function. Neurons form synapses that connect with other neurons to create neural circuits. It is believed that the efficiency of signal transmission at a synapse correlates with the volume of multiple spines present on the surface of a neuron near the synapse. For example, Non-Patent Document 1 shows that spine volume correlates with signal transmission efficiency based on experimental results showing a correlation between spine volume and the number of neurotransmitter receptors expressed on the spine. It is also known that when an animal learns, the morphology of some spines in the brain changes. Furthermore, morphological changes or abnormalities in spines have been observed in various psychiatric disorders such as Down's syndrome, depression, and fragile X syndrome (e.g., Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Matsuzaki et al.,Nature Neuroscience,2001,vol.4(11),p.1086-1092. [Non-Patent Document 2] McCann et al.,Biological psychiatry,2017,vol.82(2),p.e11-e13. Summary of the Invention [Problem to be solved by the invention]

[0004] One of the objects of the present invention is to provide an expression construct for a spine-enhancing protein that can be introduced into nerve cells to increase spine volume. [Means for solving the problem]

[0005] The present inventors focused on PSD-95 protein, which has the property of being localized in the postsynaptic membrane (posterior part) of neurons, and Kalirin-7 protein, which has the effect of increasing the volume of spines. These two proteins were co-expressed in neurons, and fused to another protein that heterodimerizes under drug administration, thereby completing the present invention.

[0006] [1] An expression construct for a spine-enhancing protein, comprising: a promoter for expression that functions in a neuronal cell; and a nucleotide sequence operably linked to the promoter, the nucleotide sequence encoding at least a domain of Kalirin-7 protein that is necessary for spine enhancement, and a nucleotide sequence encoding protein J1 that forms a heterodimer in the presence of a dimer-inducing compound. [2] An expression construct for a spine-enhancing protein, comprising: a promoter for expression that functions in nerve cells; and a nucleotide sequence operably linked to the promoter, the nucleotide sequence encoding at least a domain of the PSD-95 protein required for localization to the postsynaptic membrane, and the J2 protein that forms a heterodimer in the presence of a dimer-inducing compound. [3] The expression construct for a protein for enhancing spine according to [1], wherein the protein J1 is FKBP. [4] The expression construct of a protein for enhancing spine function described in [2], wherein the protein J2 is FRB. [5] An expression construct for a spine-enhancing protein described in [1], in which at least the domain of Kalirin-7 protein necessary for spine enhancement is encoded by the nucleotide sequence of SEQ ID NO: 1. [6] An expression construct for a spine enhancing protein described in [2], in which at least the domain of PSD-95 protein necessary for localization to the postsynaptic membrane is encoded by the nucleotide sequence of SEQ ID NO: 2. [7] The expression construct for a protein for enhancing spine function described in any one of [1] to [6], wherein the expression construct is a plasmid vector or a viral vector-based expression construct. [8] The expression construct for a spine-enhancing protein described in [7], wherein the viral vector is AAV. [9] An expression construct for a spine-enhancing protein described in any one of [1] to [8], further comprising a nucleotide sequence of a fluorescent protein operably linked to the promoter.

[10] A transformed cell into which an expression construct for the spine-enhancing protein described in any one of [1] to [9] has been genetically introduced.

[11] A transgenic non-human animal into which an expression construct for the spine-enhancing protein described in any one of [1] to [9] has been introduced.

[12] A drug for treating or preventing mental disorders, cognitive dysfunction, sleep disorders, or memory disorders, which contains as an active ingredient the expression construct described in any one of [1] to [9] and is used for the purpose of treating mental disorders, cognitive dysfunction, sleep disorders, or memory disorders in which abnormalities or changes in spine morphology are observed.

[13] A method for enhancing spines, comprising the steps of: introducing an expression construct for a spine enhancement protein described in any one of [1] to [9] into a cell or a non-human animal; and administering the dimer-inducing compound to the cell or the non-human animal. Effect of the Invention

[0007] According to the expression construct of the present invention, the volume of spines that form synapses of nerve cells can be increased by administering a drug. [Brief description of the drawings]

[0008] [Figure 1] SynCIT-Kal7 design. mVenus-FK-K7GEF translocates to spines in an A / C heterodimerizer-dependent manner, inducing spine enlargement. [Diagram 2] Example images of A / C heterodimerizer-dependent spine accumulation and expansion of mVenus-FK-K7GEF in isolated neurons. [Diagram 3] Time series data of spine accumulation and spine volume change rate of mVenus-FK-K7GEF in a representative isolated neuron. Sustained accumulation and increase are observed up to 8 hours after administration of A / C heterodimerizer. Thin lines indicate traces of individual spines in a representative neuron, and thick lines and shading indicate the mean and standard error. [Figure 4] Comparison of mVenus spine accumulation and spine volume change rate between conditions in isolated neurons. mVenus-FK-K7GEF induces spine accumulation and spine enlargement in an A / C heterodimerizer-dependent manner. mVenus-FK-K7dGEF accumulates in spines but does not induce spine enlargement. [Diagram 5] Quantitative comparison of spine accumulation and spine volume change rate of mVenus-FK-K7GEF between conditions in isolated neurons. [Figure 6] Relationship between pre-procedure spine volume and volume change rate. Spine enlargement by K7GEF tends to be greater in spines with smaller volumes. [Figure 7] The rate of spine volume change induced by various GEFs. Trio, Lfc, and Ephe1 do not induce spine enlargement. [Figure 8] Experimental diagram explaining the measurement of changes in synaptic currents by SynCIT-Kal7 manipulation. In a hippocampal slice culture system, the postsynaptic currents in CA1 cells were measured using the patch clamp method when CA3 neurons were optogenetically stimulated. "FK" in the figure is an abbreviation for "FKBP." [Figure 9] Representative traces of excitatory postsynaptic currents before and after SynCIT-Kal7 manipulation. [Figure 10] Time series data of excitatory postsynaptic currents before and after SynCIT-Kal7 manipulation. After A / C heterodimerizer, sustained enhancement of synaptic currents is observed. "Kal7" in the figure is an abbreviation of "Kalirin-7." [Figure 11] Quantitative comparison of the rate of change in excitatory postsynaptic currents by SynCIT-Kal7 manipulation between conditions. [Figure 12] Representative images of PSD95-mVenus integration by SynCIT-Kal7 manipulation. [Figure 13] Time series data of spine volume enhancement and PSD95-mVenus accumulation by SynCIT-Kal7 manipulation. "K7" in the figure is an abbreviation for "Kalirin-7." [Figure 14] Quantitative comparison of PSD95-mVenus accumulation by SynCIT-Kal7 manipulation. A / C heterodimerizer enhances PSD95-mVenus signal in spines. "GEF" in the figure stands for "Kalirin-7." [Figure 15] Time series data of spine accumulation and spine volume change rate of mVenus-FK-K7GEF under NMDA receptor inhibition [Figure 16] The average magnitude of volume change due to spontaneous spine movement was similar before and after SynCIT-Kal7 manipulation. [Figure 17] The variability in volume change due to spontaneous spine movement was similar before and after SynCIT-Kal7 manipulation. [Figure 18] Action potentials for hyperpolarizing and depolarizing currents before and after SynCIT-Kal7 manipulation. No obvious changes in membrane properties were observed upon K7GEF expression or A / C heterodimerizer administration. [Figure 19] Representative image of spine increase due to cLTP 60 min after SynCIT-Kal7 manipulation [Figure 20] Time series data of spine volume change due to cLTP after SynCIT-Kal7 manipulation [Figure 21]Comparison of spine volume changes due to cLTP between conditions. cLTP is not inhibited by SynCIT-Kal7 manipulation. [Figure 22] In vivo imaging experiment diagram [Diagram 23] Example images of A / C heterodimerizer-dependent mVenus-FK-K7GEF accumulation and expansion in spines in the living brain. [Figure 24] Time series data of spine accumulation and spine volume change rate of mVenus-FK-K7GEF in the living brain. The increase in spine volume lasts for 24 hours. mVenus-FK-K7dGEF accumulates in spines in an A / C heterodimerizer-dependent manner, but does not induce an increase in spine volume. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] <Expression construct> A first aspect of the present invention is an expression construct comprising a promoter for expression (a nucleotide sequence for initiating transcription) that functions in a neuronal cell, and a nucleotide sequence encoding a fusion protein operably linked to the promoter.

[0010] First Embodiment In a first embodiment of this aspect, the fusion protein has region A and region B in this order from the N-terminus. Region A contains at least the domain of the Kalirin-7 protein (hereinafter simply referred to as Kalirin-7) that is necessary for enhancing spine function. Region B has protein J1, one of the proteins that forms a heterodimer in the presence of a dimer-inducing compound. Within the scope of the present invention, the region A and the region B may be linked by an arbitrary linker sequence. The number of amino acid residues constituting the linker sequence may be, for example, 1 to 30. In this embodiment, the case where the domain of Kalirin-7 is contained in the N-terminal region A is described; however, conversely, the domain of Kalirin-7 may be contained in the C-terminal region B, and protein J1 may be contained in the N-terminal region A. The fusion protein may have one or more of the domains of Kalirin-7. The fusion protein may have one or more J1 proteins.

[0011] Kalirin-7 is a type of guanine nucleotide exchange factor (GEF) that acts as an activator of Rac1 and is known to rapidly migrate from dendritic shafts to nearby spines in an activity-dependent manner, contributing to spine enlargement (increase in spine volume). Therefore, at least the domain of Kalirin-7 required for spine enhancement is the domain with GEF activity (GEF domain). As shown in the experiments described below, spine enlargement does not occur even when a mutant protein lacking GEF activity is expressed.

[0012] The domain of Kalirin-7 of this embodiment preferably has an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 1. This amino acid sequence forms the DH-PH domain, which is the main part of Kalirin 7. Kalirin-7 of this embodiment may be a protein consisting of the amino acid sequence in which 1 or up to 10 amino acids are deleted, substituted or added in the amino acid sequence and having GEF activity.

[0013] In the present specification and claims, protein J1 and protein J2 are generic names for proteins that form a heterodimer between protein J1 and protein J2 in the presence of a dimer-inducing compound. Protein J1 and protein J2 do not form a homodimer by themselves.

[0014] In general, chemical inducers of dimerization (CIDs) are also called A / C heterodimerizers. CIDs are compounds that can bind to two different proteins and stabilize them in a state where they are close to each other. For example, rapamycin, a macrolide produced by actinomycetes, binds to two proteins, FKBP12 (FK506-binding protein) and FRB (FKBP-rapamycin binding protein), to form a heterodimer. In addition, when the plant hormone gibberellin binds to the receptor GID1 (Gibberellin insensitive DWARF1) protein, it causes a conformational change and binds to the GAI (Gibberellin Insensitive) protein to form a heterodimer.

[0015] In this embodiment, for example, one of protein J1 and protein J2 is FKBP12 (simply referred to as FKBP in the present specification and claims) and the other is FRB. In this case, rapamycin or a derivative thereof is used as the CID. Similarly, one of protein J1 and protein J2 is GID1 and the other is GAI. In this case, gibberellin or a derivative thereof is used as the CID.

[0016] As an example of this embodiment, when protein J1 is FKBP, the FKBP preferably has an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 3. Furthermore, the FKBP of this embodiment may be a protein consisting of the amino acid sequence in which 1 or up to 10 amino acids have been deleted, substituted or added in the amino acid sequence, and which binds to FRB in the presence of CID.

[0017] As an example of this embodiment, the fusion protein is represented as N-terminus-region A-optional linker sequence-region BC terminus. The fusion protein may have a fluorescent protein fused to the N-terminus side of region A or the C-terminus side of region B.

[0018] Examples of fluorescent proteins include mVenus, mPlum, mCherry, mStrawberry, mOrange, mKO, mCitrine, green fluorescent protein (GFP), improved GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), improved YFP (eYFP), cyan fluorescent protein (CFP), mCFPm, tdTomato, J-red, DsRed-monomer, YPet, Emerald6, CyPet, Cerulean, and T-Sapphire.

[0019] Second Embodiment In a second embodiment of this aspect, the fusion protein has region C and region D in this order from the N-terminus. Region C has the other protein, protein J2, which forms a heterodimer in the presence of a dimer-inducing compound. Region D contains at least the domain of the PSD-95 protein (hereinafter simply referred to as PSD-95) that is necessary for localization to the postsynaptic membrane. Within the scope of the present invention, the region C and the region D may be linked by an arbitrary linker sequence. The number of amino acid residues constituting the linker sequence may be, for example, 1 to 30. In this embodiment, the case where PSD-95 is contained in region D on the C-terminal side is described; however, conversely, PSD-95 may be contained in region C on the N-terminal side, and protein J2 may be contained in region D on the C-terminal side. The fusion protein may have one or more of the domains of PSD-95. The fusion protein may have one J2 protein or two or more J2 proteins. In order to increase the efficiency of forming a heterodimer with the J1 protein, the fusion protein preferably has two or three J2 proteins.

[0020] PSD-95 (postsynaptic density protein 95) is a scaffolding protein that accumulates at the site called the PSD just below the postsynaptic membrane. It is known that various receptors bind to PSD-95 and localize to the PSD. It is known that the palmitoylated region or cysteine ​​residues on the N-terminus of PSD-95, or the L27 domain, are important for the localization of the PSD.

[0021] At least the domain of PSD-95 of this embodiment that is necessary for localization to the postsynaptic membrane preferably has the above-mentioned palmitoylated region or cysteine ​​residue, or the L27 domain.

[0022] The domain of PSD-95 of this embodiment may include the PDZ domain, SH3 domain, and GK domain of wild-type PSD-95. These domains are known to interact with other proteins. In order to avoid protein-protein interactions unintended by the present inventors, the domain of PSD-95 of this embodiment preferably does not include the PDZ1 domain and the PDZ2 domain. In addition, the domain of PSD-95 of this embodiment preferably includes the PDZ3 domain, the SH3 domain, and the GK domain.

[0023] The domain of PSD-95 of this embodiment preferably has an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 2. The domain of PSD-95 of this embodiment may be a protein that consists of an amino acid sequence in which 1 or up to 10 amino acids are deleted, substituted or added in the amino acid sequence, and that is localized to PSD.

[0024] An example of the domain of PSD-95 in this embodiment is an N-terminal domain obtained by deleting a part (amino acid sequence region 65-312) of rat-derived PSD-95, and is a protein having an amino acid sequence required for localization in the PSD immediately below the postsynaptic membrane. This protein is hereinafter referred to as PSDΔ1,2.

[0025] As an example of this embodiment, when protein J2 is an FRB, the FRB preferably has an amino acid sequence encoded by the DNA sequence of SEQ ID NO: 4. Furthermore, the FRB of this embodiment may be a protein consisting of the amino acid sequence in which 1 or up to 10 amino acids have been deleted, substituted or added in the amino acid sequence, and which binds to FKBP in the presence of CID.

[0026] As an example of this embodiment, the fusion protein is represented as N-terminus-region C-optional linker sequence-region DC-terminus. The fusion protein may have a fluorescent protein fused to the N-terminus side of region C or the C-terminus side of region D. Examples of fluorescent proteins are the same as those described above.

[0027] The promoter in the expression construct of this embodiment is a nucleotide sequence that causes the fusion protein linked downstream to be expressed in nerve cells, and a promoter contained in a known vector can be applied. The promoter and the nucleotide sequence encoding the protein are "operably linked" so that the promoter can regulate the transcription and / or translation of the nucleotide sequence encoding the protein.

[0028] A promoter generally means a DNA sequence capable of regulating protein translation or RNA transcription. In this embodiment, a promoter means a transcriptional regulatory sequence capable of inducing the translation of a fusion protein of interest in eukaryotic cells, particularly in neural cells. The promoter of this embodiment can be appropriately adopted depending on the type of cells into which the expression construct is introduced, and examples thereof include the following promoters.

[0029] Examples of promoter sequences operable in eukaryotic cells include the immediate early promoter of cytomegalovirus, the early or late promoter of SV40, the tk promoter of herpes simplex virus (HSV), the adenovirus 2 major late promoter (PAdml), ​​the adenovirus 2 early promoter (PAdE2), the p19 promoter of human parvovirus associated virus (AAV), the Epstein-Barr virus (EBV) promoter, the Rous sarcoma virus (RSV) promoter, the vaccinia virus 7.5K promoter, the mouse metallothionein promoter (MT promoter), the LTR promoter of MMTV, the LTR promoter of HIV, the β-actin promoter, the EF1 alpha promoter, the promoter of the human IL-2 gene, the promoter of the human IFN gene, the promoter of the human IL-4 gene, the promoter of the human lymphotoxin gene, the promoter of the human GM-CSF gene, and promoters derived from human hemoglobin, human muscle creatine, or human metallothionein.

[0030] In the expression construct of this embodiment, a polyadenylation sequence is preferably provided as a transcription termination sequence downstream of the coding region of the fusion protein to be expressed. Examples of such known transcription termination sequences include bovine growth hormone terminator, SV40-derived polyadenylation sequence, HIV-1 polyA, β-globin polyA, HSV TK polyA, polyoma virus polyA, etc.

[0031] The expression construct of this embodiment may contain, as a selection marker, an antibiotic resistance gene commonly used by those skilled in the art, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin (G418), neomycin, and tetracycline.

[0032] The expression construct of this embodiment can be prepared in a variety of forms, and can be prepared, for example, based on a known expression vector such as a plasmid vector or a viral vector.

[0033] The expression vector is not particularly limited, and examples thereof include plasmids derived from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; plasmids derived from Bacillus subtilis such as pUB110, pTP5, and pC194; yeast-derived plasmids such as pSH19 and pSH15; bacteriophages such as λ phage; viruses such as adenovirus, adeno-associated virus (AAV), lentivirus, vaccinia virus, and baculovirus; and vectors modified from these. Among these, AAV is preferred, and AAV9, which has high tropism to the central nervous system, is more preferred.

[0034] One aspect of this aspect is an expression kit for a spine-enhancing protein, which includes the expression construct of the first embodiment of this aspect and the expression construct of the second embodiment. Here, the kit preferably includes a set of materials for expressing the spine-enhancing protein, and may include, in addition to each expression construct, various known reagents for gene transfer of each expression construct into cells or non-human cells.

[0035] Transformed cells A second aspect of the present invention is a transformed cell into which an expression construct for the spine-enhancing protein of the first aspect has been genetically introduced.

[0036] The cells to which the gene is introduced are not particularly limited as long as they are animal nerve cells, and are preferably nerve cells derived from vertebrates, particularly mammals, such as SH-SY5Y, neuro-2a, NB41A3, HCN-2, QNR / D, QNR / K2, PC-12, and RN-33B. Genetic transfer of the expression construct of the first embodiment into nerve cells can be carried out by standard methods.

[0037] The transformed cell into which the expression construct has been introduced preferably expresses at least one of the fusion proteins having Kalirin-7 and protein J1 described as the first embodiment of the first aspect and the fusion protein having PSD-95 and protein J2 described as the second embodiment of the first aspect, and preferably expresses both of the fusion proteins. By administering CID to the transformed cell expressing both fusion proteins, the fusion protein having Kalirin-7 can be accumulated in the PSD of the synapse, and the spine can be enhanced (volume increased).

[0038] <Transgenic non-human animals> A third aspect of the present invention is a transgenic non-human animal into which an expression construct for the spine-enhancing protein of the first aspect has been introduced. The non-human animal into which the gene is introduced is not particularly limited as long as it is a non-human animal, and is preferably a vertebrate, particularly a mammal, such as a mouse, rat, guinea pig, miniature pig, monkey, dog, or rabbit. Genetic transfer of the expression construct of the first aspect into a non-human animal can be carried out by a conventional method.

[0039] In the transgenic non-human animal into which the expression construct has been introduced, it is preferable that at least one of the fusion proteins having Kalirin-7 and protein J1 described as the first embodiment of the first aspect and the fusion protein having PSD-95 and protein J2 described as the second embodiment of the first aspect is expressed, particularly in the nerve cells, and it is preferable that both fusion proteins are expressed. By administering a CID to the transgenic non-human animal, the fusion protein having Kalirin-7 can be accumulated in the PSD of the synapse of the nerve cell expressing both fusion proteins, and the spine can be enhanced (the volume can be increased).

[0040] <Drugs for treating or preventing mental disorders, cognitive impairment, sleep disorders, or memory disorders> A fourth aspect of the present invention is a therapeutic or preventive agent for mental disorder, cognitive dysfunction, sleep disorder, or memory disorder, which comprises the expression construct of the first aspect as an active ingredient and is used for the purpose of treating mental disorder, cognitive dysfunction, sleep disorder, or memory disorder in which abnormality or change in spine morphology is observed. In this embodiment, the content of the expression construct of the first embodiment is preferably an amount that is therapeutically effective or prophylactically effective for each of the above-mentioned disorders to be targeted. An example of an embodiment of this aspect is a pharmaceutical composition for treating or preventing each of the above disorders, comprising a therapeutically or prophylactically effective amount of the expression construct of the first aspect and a pharma- ceutically acceptable carrier.

[0041] The pharma- ceutically acceptable carriers include those commonly used in general formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0042] The pharmaceutical composition may optionally contain, in addition to the above-mentioned components, water, pH buffer solutions, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like.

[0043] Administration of the agents and pharmaceutical compositions of this embodiment is preferably parenteral, and may utilize, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, or topical administration.

[0044] The suitable dosage of the drug and pharmaceutical composition of this embodiment varies depending on factors such as the formulation method, mode of administration, age, weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate, and reaction sensitivity, and an effective dosage for the intended treatment can usually be easily determined and prescribed by a skilled physician.

[0045] The medicament and pharmaceutical composition of this embodiment may be prepared in a unit dose form or in a multi-dose container by formulating it with a pharma- ceutically acceptable carrier and / or excipient in a manner that can be easily carried out by a person having ordinary skill in the art to which the present invention pertains. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersant or stabilizer.

[0046] The mental disorder in this embodiment may be any mental disorder in which abnormalities or changes are observed in spine morphology, and may include various mental disorders such as Down syndrome, depression, and fragile X syndrome. The cognitive dysfunction, sleep disorder, and memory disorder in this embodiment are those that are widely known in the art, and are preferably disorders in which abnormalities or changes are observed in the morphology of spines. In the nerve cells of a subject administered with the drug of this aspect, both the fusion protein having Kalirin-7 and protein J1 described as the first embodiment of the first aspect, and the fusion protein having PSD-95 and protein J2 described as the second embodiment of the first aspect are expressed. It is considered that by subsequently administering a CID to the subject, the fusion protein having Kalirin-7 is accumulated in the PSD of the synapse of the nerve cells expressing both fusion proteins, and spines are enhanced (volume is increased), thereby treating or preventing mental disorders and each of the above-mentioned disorders.

[0047] <How to strengthen spines> A fifth aspect of the present invention is a method for enhancing spines, comprising the steps of: introducing an expression construct of the spine-enhancing protein of the first aspect into a cell or a non-human animal; and administering the dimer-inducing compound (CID) to the cell or the non-human animal.

[0048] In the transfected cells or non-human animals, a first fusion protein containing Kalirin-7 and protein J1 and a second fusion protein containing PSD-95 and protein J2 are expressed. The second fusion protein is localized to the PSD of the neuron. This is due to the action of the domain for PSD localization that PSD-95 has. Meanwhile, the first fusion protein is expressed in the cytoplasm of the neuron. Gene transfection is performed by a conventional method.

[0049] CID is administered to the cells or non-human animals expressing the first and second fusion proteins in a conventional manner, and is allowed to penetrate into the neurons, thereby inducing dimerization of the proteins J1 and J2 in the neurons. As a result, the first fusion protein migrates to the PSD where the second fusion protein is localized, and the second fusion protein and the first fusion protein dimerize. As a result, the spines in which the dimerization has occurred are enhanced (volume increase, hypertrophy) due to the GEF activity of Kalirin-7, which the first fusion protein has. The enhancement of the spines means an enhancement of the synaptic transmission efficiency in the spines. EXAMPLES

[0050] All animal experiments followed the guidelines of the Animal Experimentation Committee of the Faculty of Medicine, The University of Tokyo. [DNA construct and AAV production] The constructs listed below were prepared by standard methods using PCR and In-fusion (registered trademark; Clontech). The names of each construct are listed in the following order: commercially available vector name-promoter name-protein name. The "-W" at the end stands for WPRE (woodchuck hepatitis virus posttranscriptional regulatory element).

[0051] <Expression construct> pCAG-mScarlet-W, pAAV-CaMKII(0.3)-PSD△1,2-FRBx3-W, pAAV-hSyn-PSD△1,2-FRBx3-W, pAAV-CaMKII(0.3)-mVenus-FKBP-Kalirin7DH-PH-W, pAAV-CaMKII(0.3)-mVenus-FKBP-Kalirin7DH-PH(K1393A)-W, pAAV-hSyn-DIO-mScarlet-W, pAAV-CaMKII(0.4)-Cre-W, pAAV-hSyn-DIO-PSD△1,2-FRBx3-W, pAAV-hSyn-DIO-mVenus-FKBP-Kalirin7DH-PH-W, and pAAV-hSyn-DIO-mVenus-FKBP-Kalirin7DH-PH(K1393A)-W.

[0052] Of the above constructs, the DNA sequence of "mVenus-FKBP-Kalirin7DH-PH" is SEQ ID NO:5. Of the above constructs, the DNA sequence of “PSDΔ1,2-FRBx3” is SEQ ID NO:6.

[0053] mScarlet is a red fluorescent protein. mVenus is a yellow fluorescent protein. CaMKII(0.3) is a promoter sequence of 0.3 kb obtained by deleting unnecessary parts of the calmodulin kinase II promoter. CaMKII(0.4) is a promoter sequence of 0.4 kb obtained by deleting unnecessary parts of the calmodulin kinase II promoter. hSyn is the human synapsin promoter sequence. DIO is an abbreviation for Double-Floxed Inverted Open reading frame sequence. Cre is Cre recombinase. FRBx3 is a structure in which three FRBs are linked in series with a linker (30 base pairs) consisting of 10 amino acids (the N-terminus of the second FRB is linked to the C-terminus of the first FRB via the linker). The proteins in each construct were linked via the following linkers: the DNA sequence of the linker between PSDΔ1,2 and FRB is shown in SEQ ID NO:7; the DNA sequence of the linker between mVenus and FKBP is shown in SEQ ID NO:8; and the DNA sequence of the linker between FKBP and Kalirin7DH-PH is shown in SEQ ID NO:9. Kalirin7DH-PH is a guanine nucleotide exchange factor (GEF) for rat-derived Kalirin7. K1393A in Kalirin7DH-PH(K1393A) indicates a mutant in which lysine at position 1393 is replaced by alanine.

[0054] The sequences of FRB (human origin), FKBP (human origin), PSD-95 (rat origin), and Kalirin-7 (rat origin) incorporated into each construct were cloned from cDNA. The K1393A mutation of Kalirin-7 was introduced using the following primers: 5'-gagagttacggcgtaccagctgctcctg-3' (SEQ ID NO: 10) and 5'-gtacgccgtaactctctggacagg-3' (SEQ ID NO: 11).

[0055] AAV was produced according to standard methods. Specifically, the AAV vector plasmid, pHelper (Stratagene), and pUCmini-iCAP-PHP.eB (#103005, Addgene) were transfected into HEK293 cells (AAV293, Stratagene), and after 3 days, the cells were harvested and purified twice using iodixanol. The titer of AAV was measured using qPCR.

[0056] [Assays in isolated cultures] Cortical neuronal cultures were prepared from E17 ICR mice (SLC). Cells were plated at 4.5x10 on poly-L-lysine-coated glass-bottom Petri dishes (MatTek, #1.5). 4 pieces / cm 2 The cells were seeded at a density of 100 μM. The cells were cultured in a culture medium (Neurobasal medium with B27 and L-glutamate (Gibco)) at 37°C in a humidified environment with 5% CO2. At DIV16, the cells were transfected with a mixture of three plasmids encoding the fusion proteins CAG-mScarlet, CaMKII(0.3)-PSD△1,2-FRBx3, and CaMKII(0.3)-mVenus-FKBP-Kalirin7 (GEF) using Lipofectamine 2000 (Invitrogen). After 3 to 5 days, two-color (mVenus and mScarlet) time-lapse imaging was performed every 20 min using a confocal microscope (A1R, Nikon). To induce dimerization, A / C heterodimerizer (Takara Bio) was administered at a concentration of 4 μM, and the cells were washed three times after 10 min. To analyze spine fluctuations, time-lapse imaging was performed in culture medium containing 50 μM D-APV (Abcam).

[0057] [In vivo imaging] For AAV transduction, mice were anesthetized with isoflurane (3% for induction, 1% for surgery) and fixed on a stereotaxic table (Narishige). To sparsely label mScarlet as a cytoplasmic marker, the DIO reading frame system was combined with a small amount of Cre expression. Specifically, the following AAVs were infected at a genome titer ratio of 1:900:900:15: AAVs containing four expression constructs encoding fusion proteins of CaMKII(0.4)-Cre, hSyn-DIO-mScarlet, hSyn-DIO-PSD△1,2-FRBx3, and hSyn-DIO-mVenus-FKBP-Kalirin7 (GEF) were used. After AAV injection, the incision was sealed with surgical staples and the mice were returned to their home cages and housed for approximately 4 weeks until the imaging session began. To prepare a cranial window for imaging, mice were anesthetized and head-fixed as described above. A circular bone window was created using a stainless steel trephine (φ2.3 mm; Fine Science Tools) while continuously perfusing ACSF. The bone window was covered with a cover glass (φ2.3 mm, No.0, Matsunami Glass), and the cover glass and a custom-made head plate (DMM, 3D Print) were fixed to the skull with dental cement (RelyX Unicem2 clicker, 3M). In vivo imaging was performed under 0.5% anesthesia using a confocal microscope (FVMPE-RS, Olympus) equipped with a Resonant scanner and a water-immersion objective (XLPLN25XWMP2, 25×, 1.05 NA, Olympus). Dual-color imaging (mVenus and mScarlet) was performed at 950 nm using a femtosecond pulsed laser (Insight DS+, Spectra Physics). Spine imaging positions were 60–120 μm deep from the cortical surface. 500–2000 xy images (8× digital zoom, 512 × 512 pixels) were captured with a z-axis step size of 0.02 μm.

[0058] [Measurement of changes in spine volume] Z-stack images were sequentially aligned and sum-projected using the ITK registration framework in Python. Only spines that were sufficiently far from the dendrite and continuously detected before A / C heterodimerizer injection were included in the analysis. Fluorescence intensity of the dendrite was normalized to the fluorescence of the entire imaging field. The sum of the fluorescence at the spine head was used as a proportional index of spine volume. The baseline spine volume was calculated as the average of the spine volume at all time points before A / C heterodimerizer injection. Volume change was calculated by subtracting the baseline from the spine volume at each time point. The average of the volume change at all time points after A / C heterodimerizer injection was used for statistical analysis. To estimate the absolute spine volume, Z-stack images were deconvolved using flowdec and binarized using Otsu's method in Python. In spine fluctuation analysis, the mean change in spine volume (μ) and its standard deviation (σ) were calculated every 20 minutes between 45-5 minutes before and 35-75 minutes after administration of A / C heterodimerizer.

[0059] The results are shown below. [Development of in vitro tools for enhancing synaptic strength] In the cerebral cortex, most excitatory synapses are formed in dendritic spines, and it is known that synaptic strength is significantly correlated with spine size. Furthermore, it is known that stimulation inducing long-term potentiation of synapses increases spines and increases glutamate sensitivity proportionally. Although the full picture of the molecular pathway involved in spine enlargement has not been elucidated, it has been shown that Rho family GTPases, which regulate the organization of the actin cytoskeleton, play an important role in spine remodeling. The activity of these GTPases is controlled by guanine nucleotide exchange factors (GEFs). In particular, Kalirin-7, a GEF, is known to act as an activator of Rac1 and contribute to spine enlargement by rapidly migrating from dendritic shafts to nearby spines in an activity-dependent manner.

[0060] In this example, we developed a molecular tool, SynCIT-Kal7, that artificially induces spine enlargement by mimicking the translocation of Kalirin-7 to spines (Figure 1). SynCIT-Kal7 is composed of two constructs, mVenus-FKBP-K7GEF and PSDΔ1,2-FRBx3, to induce the translocation of the GEF domain of Kalirin-7 (K7GEF) to synapses. In the former, the GEF domain of Kalirin-7 is coupled to FK506-binding protein (FKBP) and labeled with the fluorescent protein mVenus. FKBP-FRB (rapamycin binding domain of mTOR) is known as a heterodimerization system to induce specific protein-protein interactions in the presence of rapamycin and other less immunosuppressive analogs (A / C heterodimerizers). The latter construct combines FRB with a deletion mutant of PSD-95 (PSD△1,2), which accumulates in postsynapses. To prevent potential side effects associated with overexpression of PSD-95, we used PSD△1,2, which does not interact with major PDZ-binding proteins. With this design, mVenus-FKBP-K7GEF is expected to bind to PSD△1,2-FRBx3 in an A / C heterodimerizer-dependent manner and traffic to postsynapses. In fact, when SynCIT-Kal7 was expressed in the cerebral cortex and in isolated neuronal cultures, the A / C heterodimerization-dependent migration of mVenus-FK-K7GEF to synapses and the increase in spines were observed (Figs. 2-6). Kalirin-7 (dGEF), a mutant lacking GEF activity, showed synaptic migration but did not induce spine enlargement (Figs. 4 and 5), indicating that SynCIT-Kal7 induces spine enlargement dependent on GEF activity. Furthermore, spine enlargement was not induced by other GEFs, such as Trio, Lfc, and Ephexin1 sequences (Fig. 7).

[0061] To examine the changes in synaptic function associated with spine enlargement, whole-cell patch clamp recordings were performed on CA1 neurons in hippocampal slice cultures, and excitatory postsynaptic currents (EPSCs) were measured following optogenetic stimulation of CA3 axons. After administration of A / C heterodimerizer, EPSCs increased, indicating functional enhancement of synapses (Figs. 9–11). After manipulation with SynCIT-Kal7, an increase in synaptic PSD95 signals was observed in the cerebral cortex and isolated neuronal cultures, which is thought to act as a scaffold for the increase in synaptic receptors (Figs. 12–14). To examine the side effects of SynCIT-Kal7, spine fluctuations (Figs. 15–17) and membrane properties (Fig. 18) were measured, but no significant changes were observed. Furthermore, 60 min after SynCIT-Kal7, spine enlargement by chemical LTP was not inhibited (Figs. 19–21).

[0062] In vivo use of SynCIT-Kal7 To verify the applicability of SynCIT-Kal7 in the living brain, we performed time-series imaging through a skull glass window in the primary visual cortex (V1) of mice (Figure 22). SynCIT-Kal7 constructs and the fluorescent protein mScarlet were sparsely expressed in AAV by combining a double-floxed inverted open reading frame (DIO) with a low concentration of Cre. After intraperitoneal administration of A / C heterodimerizer, spine translocation of mVenus-FKBP-K7GEF and spine enlargement that lasted for 24 hours were observed by two-photon microscopy (Figures 23 and 24). Consistent with the in vitro validation, no significant spine enlargement was observed with a construct using a mutant GEF domain inactivated (dGEF) (Figure 24). Therefore, it was shown that SynCIT-Kal7 can induce spine enlargement in vivo as well. [Industrial Applicability]

[0063] The present invention is useful for functional analysis of nerve cells, and for treating or preventing mental disorders and the like.

Claims

1. A promoter for expression that functions in neural cells; A nucleotide sequence encoding at least a domain of the Kalirin-7 protein required for spine enhancement and a protein J1 that forms a heterodimer in the presence of a dimer-inducing compound, which is operably linked to the promoter; An expression construct for a protein for enhancing spine comprising:

2. A promoter for expression that functions in neural cells; An expression construct for a spine-enhancing protein, comprising a nucleotide sequence operably linked to the promoter, encoding at least a domain of the PSD-95 protein required for localization to the postsynaptic membrane, and protein J2, which forms a heterodimer in the presence of a dimer-inducing compound.

3. The expression construct of a protein for enhancing spine according to claim 1 , wherein the protein J1 is FKBP.

4. The expression construct of a spine-enhancing protein according to claim 2 , wherein the protein J2 is FRB.

5. 2. The expression construct of a protein for enhancing spines according to claim 1, wherein at least the domain of Kalirin-7 protein necessary for enhancing spines is encoded by the nucleotide sequence of SEQ ID NO:

1.

6. 3. An expression construct for a spine enhancing protein as described in claim 2, wherein at least the domain of PSD-95 protein required for localization to the postsynaptic membrane is encoded by the nucleotide sequence of SEQ ID NO:

2.

7. The expression construct of a spine enhancing protein according to claim 1 , wherein the expression construct is a plasmid vector or a viral vector-based expression construct.

8. The expression construct for a spine enhancing protein according to claim 2 , wherein the expression construct is a plasmid vector or a viral vector-based expression construct.

9. The expression construct for a spine-enhancing protein according to claim 7 , wherein the viral vector is AAV.

10. The expression construct for a spine-enhancing protein according to claim 8 , wherein the viral vector is AAV.

11. The expression construct of a spine-enhancing protein of claim 1 , further comprising a nucleotide sequence of a fluorescent protein operably linked to the promoter.

12. The expression construct of a spine-enhancing protein of claim 2 , further comprising a nucleotide sequence of a fluorescent protein operably linked to the promoter.

13. A transformed cell into which an expression construct for the spine enhancing protein according to any one of claims 1 to 12 has been genetically introduced.

14. A transgenic non-human animal into which an expression construct for the spine enhancing protein according to any one of claims 1 to 12 has been introduced.

15. A drug for treating or preventing mental disorders, cognitive dysfunction, sleep disorders, or memory disorders, comprising as an active ingredient the expression construct according to any one of claims 1 to 12, for use in treating mental disorders, cognitive dysfunction, sleep disorders, or memory disorders in which abnormalities or changes in spine morphology are observed.

16. A step of transfecting an expression construct of the spine enhancing protein according to claim 1 or 2 into a cell or a non-human animal; and a step of administering the dimer-inducing compound to the cell or the non-human animal.

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