Neurons and their applications

Abstract

To provide neurons undergoing tau aggregation and cell death; a screening kit and a screening method using the nerve cells; a drug candidate substance obtained by the screening method; human pluripotent stem cells for producing the nerve cells; and a method for producing the nerve cells.SOLUTION: Provided are neurons with introduced exogenous wild-type tau gene.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a neuronal cell that expresses tau protein, causing tau aggregation and cell death.The present invention further relates to a screening kit and screening method using the neuronal cell.The present invention further relates to a drug candidate obtained by the screening method.The present invention further relates to a method for producing human pluripotent stem cells and neuronal cells. [Background technology]

[0002] Since the onset mechanisms of neurodegenerative diseases such as Alzheimer's disease are diverse, research and development of therapeutic drugs based on various mechanisms is expected. Tau is thought to be a major factor involved in the onset of neurodegenerative diseases, and increases in the brains of patients with neurodegenerative diseases. In addition, it has been reported that there is a correlation between the amount of tau accumulation and cognitive decline, and tau is expected to be a therapeutic target for neurodegenerative diseases.

[0003] It has been reported that one of the mechanisms by which tau causes neurodegenerative diseases is that aggregated tau causes neuronal cell death. Tau aggregation progresses by polymerization of tau detached from microtubules to tau oligomers, fibrillar tau, and then to neurofibrillary tangles. However, it is still unclear how tau aggregates cause neuronal cell death in neurons, and how such neuronal cell death can be controlled. Therefore, it is expected that a neuronal cell model in which tau aggregation and neuronal cell death occur will be established, and that the model will be used to research and develop new drugs.

[0004] Non-Patent Document 1 describes a method of artificially expressing tau by introducing a tau expression vector from the outside into non-neuronal cells that do not express tau, and quantifying tau by various methods. Non-Patent Document 2 describes the induction of tau aggregation and cell death by overexpressing tau using a Tet-on AAV (adeno-associated virus) vector in human iPS cell-derived NPCs (neural progenitor cells) and adding tau aggregation seeds (aggregated tau fragments). The method described in Non-Patent Document 2 requires a long evaluation period of 30-40 days to induce differentiation while introducing a tau gene into NPCs. In addition, a large amount of AAV with a multiplicity of infection (MOI) of 100 is used.

[0005] Patent Document 1 describes the reproduction of tau aggregation and neuronal cell death using patient-derived iPS cell-derived neurons with mutant tau genes, but does not describe examples of the introduction of exogenous tau genes. In addition, it has been reported that tau aggregation and cell death are caused by mutant tau because tau aggregation and cell death have been observed in neurons with mutant tau genes. Patent Document 2 describes the introduction of exogenous mutant γPKC-GFP into CHO cells, expression control by tet-off, and evaluation of aggregation inhibition by trehalose, but does not describe examples of the introduction of exogenous tau genes. Patent Document 3 describes the use of H4-tau cells in which tau has been introduced into neuroblastoma cells and mouse neurons, in which overexpression of PRKX causes phosphorylation of tau, resulting in aggregation and cell death. The invention of Patent Document 3 is for evaluating the effect of PRKX, and tau will not aggregate without PRKX. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Comput Struct Biotechnol J. 2014 Oct 2;12(20-21):7-13 [Non-Patent Document 2] J Biomol Screen. 2016 Sep;21(8):804-15 [Patent documents]

[0007] [Patent Document 1] International Publication No. WO16 / 076435 [Patent Document 2] JP 2008-220302 A [Patent Document 3] International Publication No. WO09 / 101942 Summary of the Invention [Problem to be solved by the invention]

[0008] Known conventional methods for evaluating the aggregation or toxicity of intracellular tau include a method of overexpressing mutant tau, a method of overexpressing mutant tau and treating with an aggregation promoter (Non-Patent Documents 1 and 2), and a method of using human pluripotent stem cell-derived nerve cells established from patient cells (Patent Document 1). The method of overexpressing mutant tau has the disadvantage that it is unable to reproduce the pathology caused by the aggregation of wild-type tau. In addition, the method of using a tau aggregation promoter in combination has the disadvantage that it is difficult to evaluate the phenotype of tau alone, for example, because the tau aggregation promoter itself is toxic. In addition, the method of using human pluripotent stem cell-derived nerve cells established from patient cells generally requires long-term culture for the expression of the phenotype, making it difficult to perform drug screening.

[0009] An object of the present invention is to provide a neuronal cell that undergoes tau aggregation and cell death.A further object of the present invention is to provide a screening kit and a screening method using the above-mentioned neuronal cell.A further object of the present invention is to provide a drug candidate substance obtained by the above-mentioned screening method.A further object of the present invention is to provide a human pluripotent stem cell for producing the above-mentioned neuronal cell, and a method for producing the above-mentioned neuronal cell. [Means for solving the problem]

[0010] As a result of intensive research into solving the above problems, the present inventors have succeeded in inducing tau aggregation and cell death by introducing an exogenous wild-type tau gene into nerve cells. The present invention was completed based on the above findings.

[0011] That is, according to the present invention, the following inventions are provided. <1> Neuronal cells harboring an exogenous wild-type tau gene introduced. <2> Derived from human pluripotent stem cells, <1> A neuron according to claim 1. <3> The human pluripotent stem cells are human pluripotent stem cells derived from a healthy individual. <2> A neuron according to claim 1. <4> Exogenous wild-type tau gene is expressed following differentiation of human pluripotent stem cells into neural cells. <2> or <3> A neuron according to claim 1. <5> Expression of exogenous wild-type tau gene is condition-specifically regulated. <1> from <4> A nerve cell according to any one of the preceding claims. <6> Tau aggregates when the exogenous wild-type tau gene is overexpressed; <1> from <5> A nerve cell according to any one of the above. <7> Tau aggregation is solely due to gene expression, <1> from <6> A nerve cell according to any one of the above. <8> <1> from <7> A screening kit for a drug candidate substance that acts on changes caused by tau overexpression, comprising the nerve cell described in any one of the above. <9> <1> from <7> 2. A method for screening a drug candidate substance having an effect of inhibiting tau aggregation or cell death, using the neuronal cell according to any one of the above. <10> <9> A drug candidate substance obtained by the screening method described above. <11> A human pluripotent stem cell having an exogenous wild-type tau gene introduced therein. <12> A method for producing neural cells, comprising: introducing an exogenous wild-type tau gene into human pluripotent stem cells; and differentiating the human pluripotent stem cells into neural cells. <13> The expression of exogenous wild-type tau gene and the process of gene expression for differentiation of neural cells are the TET system. <12> The method described above. Effect of the Invention

[0012] In the neuronal cells of the present invention, tau aggregation and cell death can occur simply by expressing wild-type tau. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows the map of the CSIV-miR-9 / 9*-124-mRFP1-TRE-EF-BsdT vector. [Diagram 2] FIG. 2 shows the map of the CSII-TRE-hTau(1N4R)-IRES-Zeo vector. [Diagram 3] FIG. 3 shows a schematic diagram of a method for generating neuronal cells and overexpression of tau by lentivirus. [Figure 4] FIG. 4 shows detection of overexpressed tau by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). [Diagram 5] FIG. 5 shows neurite degeneration due to tau overexpression in hiPSC (human induced pluripotent stem cell)-derived neurons. [Figure 6] FIG. 6 shows detection of neuronal cell death due to tau overexpression. [Figure 7] FIG. 7 shows detection of aggregated tau in neurites by tau overexpression. [Figure 8] FIG. 8 shows the pharmacological effects of Tau polymerization inhibitors and microtubule stabilizing agents. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below. In this specification, "NP_", "NM_", and "NG_" and the numbers following them respectively represent the ID of the amino acid sequence (NP_~), the nucleotide sequence of the transcript (NM_~), and the genomic DNA sequence (NG_~) registered as reference sequences in the NCBI (National Center for Biotechnology Information) database.

[0015] <Nerve cells> The neuronal cells of the present invention are neuronal cells having an exogenous wild-type tau gene introduced therein. Preferably, the neural cells of the present invention are cells derived from pluripotent stem cells.

[0016] "Pluripotent stem cells" refer to cells that have both the ability to differentiate into all cells that make up a living body (pluripotency) and the ability to produce daughter cells with the same differentiation ability through cell division (self-renewal ability). Pluripotency can be evaluated by transplanting the cells to be evaluated into nude mice and testing for the presence or absence of teratoma formation containing cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0017] Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells), but are not limited thereto as long as the cells have both pluripotency and self-replication ability. Preferably, ES cells or iPS cells are used. More preferably, iPS cells are used. The pluripotent stem cells are preferably mammalian cells (e.g., primates such as humans and chimpanzees, and rodents such as mice and rats). The pluripotent stem cells are preferably human pluripotent stem cells, and more preferably human pluripotent stem cells derived from healthy subjects. In a preferred embodiment of the present invention, human iPS cells are used as the pluripotent stem cells.

[0018] ES cells can be established, for example, by culturing early pre-implantation embryos, inner cell masses constituting the above-mentioned early embryos, single blastomeres, etc. (Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, JA et al., Science, 282, 1145-1147 (1998)). As the early embryo, an early embryo produced by nuclear transfer of the nucleus of a somatic cell may be used (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), Akira Iritani et al. (Protein Nucleic Acid Enzymes, 44, 892 (1999)), Baguisi et al. (Nature Biotechnology, 17, 456 (1999)), Wakayama et al. (Nature, 394, 369 (1998); Nature Genetics, 22, 127 (1999); Proc. Natl. Acad. Sci. USA, 96, 14984 (1999)), Rideout III et al. (Nature Genetics, 24, 109 (2000), Tachibana et al. (Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer, Cell (2013) in press). Parthenogenetic embryos may be used as the initial locus (Kim et al. (Science, 315, 482-486 (2007)), Nakajima et al. (Stem Cells, 25, 983-985 (2007)), Kim et al. (Cell Stem Cell, 1, 346-352 (2007)), Revazova et al. (Cloning Stem Cells, 9, 432-449 (2007)), Revazova et al. (Cloning Stem Cells, 10, 11-24 (2008)).In addition to the above-mentioned papers, for the production of ES cells, references include Strelchenko N., et al. Reprod Biomed Online. 9: 623-629, 2004; Klimanskaya I., et al. Nature 444: 481-485, 2006; Chung Y., et al. Cell Stem Cell 2: 113-117, 2008; Zhang X., et al. Stem Cells 24: 2669-2676, 2006; Wassarman, PM et al. Methods in Enzymology, Vol. 365, 2003, etc. It should be noted that fused ES cells obtained by cell fusion between ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

[0019] Some ES cells are available from collections or commercially available, for example, human ES cells are available from the Institute for Frontier Medical Sciences, Kyoto University (e.g., KhES-1, KhES-2, and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0020] EG cells can be established by culturing primordial germ cells in the presence of LIF (leukemia inhibitory factor), bFGF (basic fibroblast growth factor), and SCF (stem cell factor), for example (Matsui et al., Cell, 70, 841-847 (1992); Shamblott et al., Proc. Natl. Acad. Sci. USA, 95 (23), 13726-13731 (1998); Turnpenny et al., Stem Cells, 21 (5), 598-609, (2003)).

[0021] "Induced pluripotent stem cells (iPS cells)" are cells that have pluripotency (multi-differentiation ability) and proliferation ability, and are produced by reprogramming somatic cells by introducing reprogramming factors. Induced pluripotent stem cells exhibit properties similar to ES cells. The somatic cells used to produce iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. In addition, their origin is also not particularly limited, but somatic cells of mammals (e.g., primates such as humans and chimpanzees, and rodents such as mice and rats), and particularly preferably human somatic cells, are used. iPS cells can be produced by various methods reported so far. In addition, it is naturally assumed that iPS cell production methods developed in the future will also be applied.

[0022] The most basic method for generating iPS cells is to introduce the four transcription factors Oct3 / 4, Sox2, Klf4, and c-Myc into cells using a virus (Takahashi K, Yamanaka S: Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131(5), 861-72, 2007). There is a report on the establishment of human iPS cells by introducing the four factors Oct4, Sox2, Lin28, and Nonog (Yu J, et al: Science 318(5858), 1917-1920, 2007). It has also been reported that iPS cells can be established by introducing three factors other than c-Myc (Nakagawa M, et al: Nat. Biotechnol. 26(1), 101-106, 2008), two factors Oct3 / 4 and Klf4 (Kim JB, et al: Nature 454(7204), 646-650, 2008), or only Oct3 / 4 (Kim JB, et al: Cell 136(3), 411-419, 2009). In addition, a method of introducing a protein, which is the product of gene expression, into cells has also been reported (Zhou H, Wu S, Joo JY, et al: Cell Stem Cell 4, 381-384, 2009; Kim D, Kim CH, Moon JI, et al: Cell Stem Cell 4, 472-476, 2009). On the other hand, it has been reported that the use of histone methyltransferase G9a inhibitor BIX-01294, histone deacetylase inhibitor valproic acid (VPA) or BayK8644, etc., can improve the production efficiency and reduce the amount of factors introduced (Huangfu D, et al: Nat. Biotechnol. 26(7), 795-797, 2008; Huangfu D, et al: Nat. Biotechnol. 26(11), 1269-1275, 2008; Silva J, et al: PLoS. Biol. 6(10), e253, 2008).Methods for gene transfer have also been investigated, and in addition to retroviruses, lentiviruses (Yu J, et al: Science 318(5858), 1917-1920, 2007), adenoviruses (Stadtfeld M, et al: Science 322(5903), 945-949, 2008), plasmids (Okita K, et al: Science322(5903), 949-953, 2008), transposon vectors (Woltjen K, Michael IP,Mohseni P, et al: Nature458,766-770, 2009;Kaji K,Norrby K, Pac a A,et al:Nature458,771-775, 2009;Yusa K, Rad R,Takeda J, et al:Nat Methods 6, 363-369, 2009) or episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) have been developed for gene transfer.

[0023] Cells that have been transformed into iPS cells, i.e., reprogrammed, can be selected using the expression of pluripotent stem cell markers (undifferentiated markers) such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1, and Cript as indicators. The selected cells are collected as iPS cells.

[0024] iPS cells can also be obtained from, for example, FUJIFILM Cellular Dynamics, Inc., Kyoto University, or the RIKEN BioResource Center.

[0025] Pluripotent stem cells can be cultured using a medium suitable for culturing pluripotent stem cells. When iPS cells are used as pluripotent stem cells, for example, StemFit (registered trademark) AK02N (Ajinomoto), mTeSR (registered trademark) 1 (Stemcell Technologies), StemFlex (registered trademark), etc. can be used. The culture may be performed on a plate (for example, a 6-well plate, etc.) or in a flask, but is preferably performed on a plate. The culture period is not particularly limited, and the culture can be performed for, for example, 1 to 7 days.

[0026] The neuronal cells of the present invention have an exogenous wild-type tau gene introduced from outside. Tau (also known as microtubule-associated protein tau, MAPT) is a protein encoded by the MAPT gene (Official full name: microtubule-associated protein tau, Official symbol; MAPT, NG_007398.1) located on chromosome 17 (17q21.1) in humans, and six isoforms generated by alternative splicing have been identified.

[0027] Each isoform differs in the number of characteristic 29 amino acid sequences (N) on the N-terminus (0-2 repeats) and the number of repeats (R) involved in microtubule binding on the C-terminus (3 or 4 repeats). Depending on the number of these sequences, they are classified as 0N3R type (352 amino acids, NP_058525.1, NM_016841.4), 1N3R type (381 amino acids, NP_001190180.1, NM_001203251.1), 2N3R type (410 amino acids, NP_001190181.1, NM_001203251.1), and 3N3R type (420 amino acids, NP_001190181.1, NM_001203251.1). They are classified into types 0N4R (383 amino acids, NP_058518.1, NM_016834.4), 0N4R (412 amino acids, NP_001116539.1, NM_001123067.3), and 2N4R (441 amino acids, NP_005901.2, NM_005910.5) (in parentheses, the number of amino acid residues of each human isoform, the ID of the standard amino acid sequence, and the ID of the standard nucleotide sequence of the transcript are shown as examples).

[0028] The tau gene in the present invention may be a tau gene of a mammal other than a human (e.g., a rodent such as a mouse or a rat, or a primate such as a marmoset). There are six types of tau protein isoforms that may be generated by alternative splicing in the brain of a living mammal, namely, the 0N3R type, 1N3R type, 2N3R type, 0N4R type, 1N4R type, and 2N4R type. Only 3R tau is expressed in the human fetal brain, but all six types are expressed in the adult human brain, and in normal individuals, the expression ratio of 4R (4-repeat tau) to 3R (3-repeat tau) (= 4-repeat tau / 3-repeat tau) is similar. In mice, only 3R tau is expressed until neonatal age, but after weaning, only 4R tau is expressed. In rats and marmosets, only 3R tau is expressed in the brain until neonatal age, but after weaning, only 4R tau is expressed. In the present invention, unless otherwise specified, tau may be either 3-repeat tau or 4-repeat tau.

[0029] <Manufacturing nerve cells> The neural cells of the present invention can be produced by differentiating a human pluripotent stem cell having an exogenous wild-type tau gene introduced thereinto into a neural cell. The human pluripotent stem cell having an exogenous wild-type tau gene introduced thereinto can be produced by introducing an exogenous wild-type tau gene into a human pluripotent stem cell. That is, according to the present invention, there is provided a method for producing neural cells, which comprises introducing an exogenous wild-type tau gene into a human pluripotent stem cell and differentiating the human pluripotent stem cell into a neural cell.

[0030] Methods for inducing differentiation of pluripotent stem cells into neurons include, but are not limited to, a method in which neural stem cells are produced from pluripotent stem cells using treatment with a low molecular weight compound, etc., and then the neural cells are induced, and a method in which the neural cells are directly induced by gene expression, etc. In the present invention, since the differentiation of pluripotent stem cells into neurons is induced, neural cells that do not contain glial cells can be produced. The neural cells of the present invention are preferably a cell population that does not contain glial cells. Furthermore, the nerve cells of the present invention are preferably not three-dimensionally cultured cells, and are more preferably monolayer cultured cells. Methods for inducing differentiation of pluripotent stem cells into neural cells include, for example, (1) A method in which embryoid bodies (cell masses containing neural progenitor cells) are formed and differentiated in a serum-free medium (SFEB method: Watanabe K., et al., Nat. Neurosci., 8: 288-296, 2005; SFEBq method: Wataya T., et al. Proc. Natl. Acad. Sci. USA., 105: 11796-11801, 2008); (2) A method of culturing and differentiating on stromal cells (SDIA method: Kawasaki H., et al., Neuron, 28:31-40, 2000); (3) A method of culturing and differentiating on drug-added Matrigel (Chambers SM, et al, Nat. Biotechnol., 27:275-280, 2009); (4) A method of differentiation by culturing in a medium containing a low molecular weight compound as an alternative to cytokines (U.S. Patent No. 5,843,780); (5) A method of differentiating pluripotent stem cells by introducing and expressing neural induction factors (such as neurogenin2 (Ngn2)) (WO2014 / 148646; and Zhang Y., et al, Neuron, 78:785-98, 2013); (6) A method of inducing differentiation by introducing and expressing miR-9 / 9*-124 into pluripotent stem cells; and combinations of these methods.

[0031] Among the above, (5) the method of introducing and expressing neurogenin2 into pluripotent stem cells is preferred because it allows for the production of mature nerve cells in a short period of time and with high efficiency. Furthermore, (6) the method of introducing and expressing miR-9 / 9*-124 into pluripotent stem cells to cause differentiation is also preferred. As a method for inducing differentiation of pluripotent stem cells into nerve cells, a preferred method is a method of directly inducing nerve cells by expressing Ngn2 alone or Ngn2 and miR-9 / 9*-124. Most preferred is a method of inducing nerve cells by expressing Ngn2 alone or Ngn2 + miR-9 / 9*-124 using the TET-on promoter.

[0032] Neurogenin2 protein is a transcription factor known to promote differentiation into nerve cells during development, and its amino acid sequence is exemplified by NP_076924 in humans and NP_033848 in mice. Neurogenin2 gene (official full name: neurogenin2, official symbol: NEUROG2, also called Ngn2 gene) is DNA encoding Neurogenin2 protein, and examples thereof include DNA having the nucleotide sequence of NM_009718 (mouse) or NM_024019 (human) registered as a standard sequence, or a transcript variant thereof. In addition, it may be DNA having a degree of complementarity that allows hybridization under stringent conditions to nucleic acids having the above-mentioned standard sequence and the sequence of a transcript variant.

[0033] Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe is bound. For example, washing conditions after hybridization can usually be about "1xSSC (Saline Sodium Citrate Buffer), 0.1% SDS, 37°C". It is preferable that the complementary strand maintains a hybridized state with the target positive strand even when washed under such conditions. As more stringent hybridization conditions, washing conditions of about "0.5xSSC, 0.1% SDS, 42°C" can be mentioned, and even more stringent conditions can be mentioned such that the positive strand and complementary strand maintain a hybridized state even when washed under washing conditions of about "0.1xSSC, 0.1% SDS, 65°C". Specifically, examples of such complementary strands include a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and a strand consisting of a base sequence that has an identity of at least 90%, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more with the above strand.

[0034] Expression of Neurogenin 2 in pluripotent stem cells can be carried out by introducing a nucleic acid (DNA or RNA) encoding Neurogenin 2 or Neurogenin 2 (protein) into the pluripotent stem cells. Expression of miR-9 / 9*-124 in pluripotent stem cells can be carried out by introducing a nucleic acid (DNA or RNA) encoding miR-9 / 9*-124 into the pluripotent stem cells.

[0035] In the present invention, when a nucleic acid encoding Neurogenin2 and a nucleic acid encoding miR-9 / 9*-124 are introduced into a cell, for example, vectors such as viruses, plasmids, and artificial chromosomes can be introduced into pluripotent stem cells using techniques such as lipofection, liposomes, and microinjection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and Sendai viral vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Mammalian plasmids can be used as plasmids. The vector may contain a control sequence (promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc.) for expressing the Neurogenin2 protein or miR-9 / 9*-124, and may further contain, as necessary, a drug resistance gene (e.g., kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), a selection marker sequence such as a thymidine kinase gene or a diphtheria toxin gene, a reporter gene sequence such as β-glucuronidase (GUS) or FLAG, etc. In particular, it is preferable that a nucleotide sequence encoding the amino acid sequence of a protein is functionally linked to an inducible promoter sequence so that the expression of the Neurogenin2 protein or miR-9 / 9*-124 can be rapidly induced at a desired time.

[0036] Examples of inducible promoters include drug-responsive promoters, and a preferred example thereof is a tetracycline-responsive promoter (CMV minimal promoter having a tetracycline-responsive sequence (TRE) with seven consecutive tetO sequences). For example, the Tet-On / Off Advanced expression induction system is exemplified, but since it is desirable to express the corresponding gene in the presence of tetracycline, the Tet-On system is preferred. That is, it is a system that simultaneously expresses reverse tetR (rtetR) and a fusion protein (rtTA) with VP16AD. The Tet-On system can be obtained from Clontech and used. In addition, cumate-responsive promoters (Q-mate system, Krackeler Scientific, National Research Council (NRC), etc.), estrogen-responsive promoters (WO2006 / 129735, and GenoStat inducible expression system, Upstate cell signaling solutions), RSL1-responsive promoters (RheoSwitch mammalian inducible expression system, New England Biolabs), etc. can also be preferably used. Among these, tetracycline-responsive promoters and cumate-responsive promoters are particularly preferred because of their high specificity to expression inducers and low toxicity, and tetracycline-responsive promoters are the most preferred.

[0037] When a cumate-responsive promoter is used, it is preferable that it also has a pattern for expressing the CymR repressor in pluripotent stem cells. In addition, the control sequence and regulators of the above promoter (rtTA and / or CymR repressor, etc.) may be supplied by a vector into which the neurogenin2 gene or miR-9 / 9*-124 has been introduced.

[0038] When a tetracycline-responsive promoter is used, the expression of Neurogenin2 or miR-9 / 9*-124 can be maintained by continuously adding tetracycline or its derivative doxycycline (hereinafter abbreviated as DOX) to the medium for a desired period of time. When a cumate-responsive promoter is used, the expression of Neurogenin2 or miR-9 / 9*-124 can be maintained by continuously adding cumate to the medium for a desired period of time.

[0039] As a promoter for expressing Neurogenin2 protein or miR-9 / 9*-124, a constitutive expression promoter (such as a cytomegalovirus (CMV)-derived promoter, an EF1 promoter, a ubiquitin C (UbC) promoter, or a murine stem cell virus (MSCV)-derived promoter) or a neuron-specific promoter (such as the Syn promoter) may be used.

[0040] When a drug-responsive promoter is used, the expression of Neurogenin2 or miR-9 / 9*-124 can also be stopped by removing the corresponding drug from the medium (for example, by replacing the medium with one that does not contain the drug).

[0041] In the present invention, when a nucleic acid encoding Neurogenin2 or miR-9 / 9*-124 is introduced in the form of RNA, it may be introduced into pluripotent stem cells by techniques such as electroporation, lipofection, microinjection, etc. To maintain the expression of Neurogenin2 or miR-9 / 9*-124 in the cells, the introduction may be carried out multiple times, for example, two, three, four, or five times.

[0042] In the present invention, when Neurogenin2 is introduced in the form of a protein, it may be introduced into pluripotent stem cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (e.g., HIV-derived TAT and polyarginine), microinjection, etc. To maintain the expression of Neurogenin2 in the cells, it may be introduced multiple times, for example, two, three, four, or five times.

[0043] In the present invention, there is no particular upper limit to the period during which Neurogenin2 or miR-9 / 9*-124 is expressed in pluripotent stem cells for the induction of neural cells, but in the case of human pluripotent stem cells, the period is at least 2 days, preferably at least 3 days, and more preferably at least 4 days.

[0044] After induction of expression in the method of differentiation by introducing and expressing Neurogenin2 or miR-9 / 9*-124, it is preferable to culture the pluripotent stem cells in a medium suitable for inducing differentiation into neural cells (called neural differentiation medium). As such a medium, a basal medium alone or a basal medium supplemented with a neurotrophic factor can be used. The neurotrophic factor in the present invention is a ligand of a membrane receptor that plays an important role in the survival and functional maintenance of nerve cells, and examples thereof include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 / 5 (NT-4 / 5), neurotrophin 6 (NT-6), basic FGF, acidic FGF, FGF-5, epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin, insulin-like growth factor 1 (IGF1), insulin-like growth factor 2 (IGF 2), Glia cell line-derived neurotrophic factor (GDNF), transforming growth factor-b2, transforming growth factor-b3, interleukin 6 (IL-6), ciliary neurotrophic factor (CNTF), and LIF. Among these, preferred neurotrophic factors are GDNF, BDNF, and / or NT-3.

[0045] Examples of basal media include Glasgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 (F12) medium, Dulbecco's Modified Eagle Medium:Nutrient Mixture F-12 (DMEM / F-12) medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium medium (Lifetechnologies), Neurobasal Plus Medium medium (Thermo Fisher Scientific), and Brainphys (STEMCELL Technologies), as well as mixtures thereof.

[0046] The basal medium may contain serum or may be serum-free. If necessary, the medium may contain one or more serum substitutes, such as Knockout Serum Replacement (KSR) (a serum substitute for FBS during ES cell culture), N2 supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), B27 Plus supplement (Thermo Fisher Scientific), Culture One supplement (Thermo Fisher Scientific), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, and may also contain one or more substances, such as lipids, amino acids, L-glutamine, Glutamax (Thermo Fisher Scientific), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, selenate, progesterone, and putrescine.

[0047] In the present invention, the medium for neural differentiation is Neurobasal plus Medium, supplemented with B27 Plus supplement, Culture One supplement, Glutamax, dbcAMP, L-ascorbic acid, Y27632, and DAPT. It is particularly preferable to use a medium supplemented with (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester).

[0048] The culture temperature when inducing differentiation of neural cells is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0049] In the present invention, a neuron is preferably a cell that expresses at least one of the neuron-specific marker genes, which are composed of β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2), and has β-III tubulin-positive processes (hereinafter referred to as neurites).

[0050] In the present invention, more preferred nerve cells are morphologically mature nerve cells, and even more preferred are glutamatergic nerve cells. Here, the term "morphologically mature nerve cells" refers to nerve cells with thickened cell bodies and sufficiently extended neurites (as a guide, the neurite length is about 5 times or more the diameter of the cell body).

[0051] The neural cells of the present invention can be produced by introducing an exogenous wild-type tau gene into neural cells induced to differentiate from pluripotent stem cells as described above. Alternatively, the neural cells of the present invention may be produced by introducing an exogenous wild-type tau gene into pluripotent stem cells before they are induced to differentiate into neural cells, and then inducing the differentiation of the pluripotent stem cells into neural cells.

[0052] Methods for introducing an exogenous wild-type tau gene include a viral vector, lipofection, electroporation, etc. A viral vector is preferred, and lentivirus, adeno-associated virus (AAV), Sendai virus, retrovirus, etc. are more preferred, and lentivirus is most preferred.

[0053] For the overexpression of exogenous wild-type tau, a promoter is preferably used. Preferably, a constitutive expression promoter (such as a cytomegalovirus (CMV)-derived promoter, an EF1 promoter, a ubiquitin C (UbC) promoter, or a murine stem cell virus (MSCV)-derived promoter), a neuron-specific promoter (such as a Syn promoter), or a drug-inducible promoter (such as TET-on, TET-off, or isopropyl-β-thiogalactopyranoside (IPTG)), or the like, may be used, and more preferably, a TET-on promoter may be used.

[0054] In the neuronal cells of the present invention, an exogenous wild-type tau gene is preferably expressed after differentiation from human pluripotent stem cells into neurons, and the expression of the exogenous wild-type tau gene is preferably controlled in a condition-specific manner.

[0055] In the present invention, it is particularly preferred that the process of expression of an exogenous wild-type tau gene and gene expression for differentiation of nerve cells is a TET system (TET-on system or TET-off system), which is a system that can reversibly regulate the expression of a target gene in a cell by administration of doxycycline, a tetracycline derivative.

[0056] In the neuronal cells of the present invention, tau aggregates when an exogenous wild-type tau gene is overexpressed. It is preferable that tau aggregation is caused only by the expression of an exogenous wild-type tau gene.

[0057] In one preferred embodiment of the present invention, human neurons can be produced from human pluripotent stem cells. Preferably, neurons are produced by TET-on-inducible expression of Ngn2 and miR-9 / 9*-124. Preferably, the produced neurons are frozen and stored, and used in a hypnotic state during evaluation. The produced neurons are preferably seeded on a coated cell culture plate, and more preferably, the neurons are seeded in a monolayer. After seeding the neurons, a tau gene can be exogenously introduced to express tau. Particularly preferably, a lentivirus vector can be used to express tau in a TET-on-inducible manner by doxycycline treatment.

[0058] <Screening method> The present invention relates to a screening kit for a drug candidate substance having an effect of acting on changes due to tau overexpression, comprising the above-mentioned neuronal cell of the present invention, and a screening method for a drug candidate substance having an effect of inhibiting tau aggregation or cell death, using the above-mentioned neuronal cell of the present invention.The present invention further relates to a drug candidate substance obtained by the above-mentioned screening method.

[0059] The neuronal cells of the present invention can induce tau aggregation and neuronal cell death by overexpressing tau, and therefore can be used for screening of drug candidates for central nervous system diseases and analysis of pathological mechanisms. That is, in the neuronal cells of the present invention, tau aggregation and the accompanying neuronal cell death can be manifested only by expressing wild-type tau. Furthermore, drug candidate substances can be screened using these phenotypes as indicators. In the present invention, not only neuronal cell death but also fragmentation and disappearance of neurites, increase in aggregated tau, and increase in phosphorylated tau can be obtained. Furthermore, the response of drugs to these phenotypes can be evaluated.

[0060] In the present invention, after overexpression of tau in the neurons of the present invention, tau aggregation, phosphorylation, tau amount, tau localization, etc. can be evaluated by live or dead cell evaluation, Western blotting, ELISA (enzyme-linked immunosorbent assay), or biochemical analysis by staining, and further neurites, various nerve-related proteins, disease markers, etc. can be evaluated. In one embodiment of the present invention, tau can be aggregated and cell death can be induced without any other tau aggregation-promoting manipulation other than overexpression of wild-type tau, such as the use of aggregates of tau fragments (e.g., K18), dephosphorylation inhibitors (e.g., okadaic acid), and protein aggregation promoters (e.g., Congo Red). In the present invention, overexpression means that the target mRNA or protein is expressed in a greater amount than in the absence of manipulation, preferably at an equal or greater level than the endogenous protein.

[0061] In the present invention, for example, (1) contacting the neuronal cell of the present invention with a test substance; (2) overexpressing tau in the neuronal cell; (3) measuring tau aggregation or associated neuronal cell death in the neuronal cells; and (4) selecting the test substance as a preventive or therapeutic drug for tauopathy when the tau aggregation or associated neuronal death measured in step (3) in the case where the test substance is contacted in step (1) is lower than the tau aggregation or associated neuronal death in the case where the test substance is not contacted in step (1); The present invention provides a method for screening for a prophylactic or therapeutic agent for tauopathy, comprising:

[0062] The above step (1) is a step of seeding nerve cells on a culture plate to which a culture medium containing a test substance has been added, or adding the test substance after seeding nerve cells on a culture plate. The test substance may be added before or after a treatment for overexpressing exogenous tau. Preferably, the test substance is added before overexpressing tau.

[0063] In the above step (2), a culture medium is added to a culture plate, and then a treatment for overexpressing tau (such as adding a lentivirus) is performed, and then the neurons are seeded, or a treatment for overexpressing tau is performed after the neurons are seeded. Preferably, tau is overexpressed after the neurons are seeded. More preferably, tau is overexpressed two days after the neurons are seeded.

[0064] In the above step (3), neuronal cell death can be detected using a live cell detection reagent such as Cell Titer Glo (Promega) or Cell counting kit-8 (Dojindo), or a cell death detection reagent such as Propidium Iodide or NucGreen Dead (Thermo Fisher Scientific). Preferably, Cell Titer Glo is used. Tau aggregation can be detected by fixing the neuronal cells by treating them with paraformaldehyde, neutral buffered formalin, ethanol, or the like, and then performing fluorescent staining using an antibody (tau antibody, tau oligomer antibody, βIII tubulin antibody, or the like). Alternatively, proteins can be recovered and electrophoresed by native PAGE, non-reduced SDS-PAGE, SDS-PAGE, or the like, and then detected using Coomassie Brilliant Blue staining or a tau antibody. Preferably, after fixation with paraformaldehyde, visualization is performed by fluorescent staining using a tau antibody or a tau oligomer antibody. After visualization, images are taken and analyzed using a fluorescent imaging device (In Cell Analyzer (GE Healthcare), IncuCyte (Sartorius), CQ-1 (Yokogawa Electric)). Preferably, an In Cell Analyzer is used. More preferably, an In Cell Analyzer 6000 is used.

[0065] The analysis is performed by measuring the neurite length or fluorescence intensity of tau oligomer antibody positive. By using an antibody that recognizes the overexpressed tau isoform (4R tau antibody when 1N4R tau is overexpressed in nerve cells where 4R tau is hardly expressed), exogenous tau is introduced and overexpressed neurites can be detected and the neurite length can be measured. This indicator can be used as an internal standard for overexpression. Preferably, the value obtained by correcting the tau oligomer antibody positive neurite length with the 4R tau antibody positive neurite length can be used as an indicator of tau aggregation.

[0066] In step (4), the term "reduced compared to tau aggregation in the absence of contact with the test substance in step (1)" is not particularly limited as long as it is low, but a test substance that has the effect of reducing tau aggregation by contact with the test substance to preferably 80% or less, more preferably 50% or less, when the tau aggregation in the absence of contact with the test substance is taken as 100%, is selected. In addition, the term "reduced compared to neuronal death in the absence of contact with the test substance in step (1)" is not particularly limited as long as it is low, but a test substance that has the effect of increasing cell viability by preferably 20% or more, more preferably 50% or more, when the cell viability in the absence of tau overexpression is taken as 100% and the cell viability in the absence of tau overexpression but contact with the test substance is taken as 0%, is selected as a test substance that reduces neuronal death.

[0067] The screening method of the present invention is useful for screening compounds or lead compounds that can be preventive or therapeutic agents for tauopathy. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal dementia (FTD), Pick's disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain dementia, neurofibrillary tangle dementia, diffuse neurofibrillary tangle disease with calcification (DNTC), and primary age-related tauopathy (PART).

[0068] Examples of test substances include proteins, peptides, antibodies, nucleic acids (gene expression vectors, siRNA, antisense oligonucleotides, mRNA), viral vectors (AAV, lentivirus, adenovirus, etc.), non-peptide compounds, synthetic compounds, synthetic low molecular weight compounds, natural compounds, cell extracts, plant extracts, animal tissue extracts, plasma, extracts derived from marine organisms, cell culture supernatants, and microbial fermentation products.

[0069] Test substances can also be obtained using any of the many approaches in combinatorial library methods known in the art, including (1) biological library methods, (2) synthetic library methods using deconvolution, (3) one-bead one-compound library methods, and (4) synthetic library methods using affinity chromatography selection. The biological library methods using affinity chromatography selection are limited to peptide libraries, while other approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12: 145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. al. (1994) J.Med.Chem.37:1233-51).Compound libraries can be produced in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9) or phages (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6). al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application Publication No. 2002 / 0103360).

[0070] In the present invention, contacting a test substance with nerve cells may be performed by adding the test substance to a culture solution for nerve cells. The contact time is not particularly limited as long as the change in the indicator can be confirmed, and may be, for example, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. The concentration of the test substance to be added can be appropriately adjusted depending on the type of compound (solubility, toxicity, etc.).

[0071] The culture medium for neurons used when contacting the test substance with the neurons is not particularly limited as long as it is a medium capable of culturing neurons, and examples thereof include the above-mentioned neuronal differentiation medium.

[0072] The culture temperature when contacting the test substance with the nerve cells is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and culture is performed in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0073] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0074] (1) Construction of lentiviral vector Lentivirus was produced according to the method of non-patent literature (Mitsuru Ishikawa, et al., Cells 2020, 9, 532;). Briefly, lentivirus was produced by transfecting HEK293T cells with three types of plasmids, namely, packaging construct (pCAG-HIVgp), VSV-G and Rev expression construct (pCMV-VSV-G-RSV-Rev), and self-inactivating (SIN) lentivirus vector construct (CSIV-miR-9 / 9*-124-mRFP1-TRE-EF-BsdT, or CSII-TRE-hTau(1N4R)-IRES-Zeo), using polyethyleneimine (Polysciences). Furthermore, the culture supernatant was concentrated by ultracentrifugation to concentrate the lentivirus. After concentration, the titer was measured using lenti-Gostix PLUS (Takara Bio) and used in the experiment. CSIV-miR-9 / 9*-124-mRFP1-TRE-EF-BsdT (Figure 1) and CSII-TRE-hTau(1N4R)-IRES-Zeo (Figure 2) are shown. For CSII-TRE-hTau(1N4R)-IRES-Zeo, we first designed primers containing attL1 and attL2 and the sequences at both ends of the tau gene sequence, and performed PCR using the tau gene as a template to create a PCR product of the tau gene with attL1 and attL2 at both ends. Then, to create a plasmid using the Gateway method, we performed reverse PCR using the pDONR (Thermo Fisher Scientific) vector and the above PCR product as a template to create a pDONR vector containing the tau gene. Then, we recombined the genes of that vector and the CSII vector using the Gateway method to create CSII-TRE-hTau(1N4R)-IRES-Zeo.

[0075] (2) Introduction of TET-on inducible Ngn2 and miR-9 / 9*-124 vectors into iPS cells According to the method of non-patent literature (Mitsuru Ishikawa, et al., Cells 2020, 9, 532;), a transposase vector (pCMV-HyPBase-PGK-Puro), a rtTA vector (PB-CAGrtTA3G-IH), and a Neurogenin2 (Ngn2) vector (PB-TET-PH-lox66FRT-NEUROG2) as shown in FIG. 3 were prepared. These vectors were introduced into iPS cells cultured with StemFit (registered trademark) by lipofection using GeneJuice (Novagen), and further, a lentivirus (CSIV-miR-9 / 9*-124-mRFP1-TRE-EF-BsdT) containing the miR-9 / 9*-124 gene was introduced into the iPS cells. Then, an iPS cell line into which the vector was stably introduced was obtained by drug selection using Puromycin, Hygromycin, and Blasticidin S.

[0076] (3) Creation of neural cells iPS cells transfected with Ngn2 and miR-9 / 9*-124 genes were detached using TrypLE select and seeded on 6-well plates coated with polyornithine and iMatrix (Nippi). The cells were incubated in a neural induction medium containing doxycycline (DOX) (Neurobasal plus medium with 2% B27 Plus supplement, 1% Culture One supplement, 200 μmol / L dbcAMP, 200 μmol / L L-ascorbic acid, 10 μmol / L Y27632, and 10 μmol / L DAPT). (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester), supplemented with 4μg / ml Dox) for 5 days to induce differentiation into neurons. Neurons were detached using TrypLE Select and frozen at Stem Cell Banker.

[0077] (4) Lentiviral vector infection and tau overexpression in iPS cell-derived neurons Frozen neurons were thawed in a water bath and plated onto a 96-well plate coated with Poly-D-lysine (PDL) and iMatrix (Nippi) at approximately 3 × 10 4 Approximately 30 × 10 cells / well or 12-well plate 4 The cells were seeded in a neural reseeding medium containing DOX (Neurobasal plus:DMEM / F12 HAM=1:1 medium, 2% B27 Plus supplement, 1% Culture One supplement, 200μmol / L dbcAMP, 200μmol / L L-ascorbic acid, 2μg / ml Dox) at a cell number of 100 cells / well. In addition, 1μmol / L Tau Accell siRNA (Dharmacon, #A-012488-13) described in (7) below, or a compound described in (9) below was added. The cells were cultured for 2 days in a 37℃ CO2 incubator, treated with lentivirus vector, and cultured for about 7 days, after which various evaluations were performed.

[0078] (5) Detection of overexpressed tau in hiPSC-derived neurons Neurons were infected with tau-expressing lentivirus vectors by the method described in (4), and after 5 days of culture, the neurons were solubilized using RIPA buffer. SDS-PAGE was performed using NuPAGE gel (Thermo Fisher Scientific). After electrophoresis, the proteins on the gel were transferred to a PVDF (polyvinylidene fluoride) membrane, blocked, and reacted with tau antibody (DAKO, 2000-fold dilution, 4°C, overnight) and secondary antibody anti-rabbit 800 nm (purchased from LI-COR, 2000-fold dilution, room temperature, 1 hour), and fluorescence was detected with Odyssey CLx (LI-COR). The results are shown in Figure 2. It was confirmed that tau was induced by overexpression (OE) in an amount greater than that of endogenous tau.

[0079] (6) Neurite degeneration due to overexpression of tau in hiPSC-derived neurons Neurons were infected with tau-expressing lentiviral vectors using the method described in (4), and 6 days later, the cells were fixed with 4% paraformaldehyde. After blocking, the cells were reacted with a primary antibody (1000-fold dilution, 4°C, overnight) and a secondary antibody (2000-fold dilution, room temperature, 1 hour), and the fluorescence was detected using a fluorescent microscope. The results are shown in Figure 3. Overexpression of 1N4R tau increased the signals of 4R tau and phosphorylated tau (AT-8, PHF-1). Furthermore, overexpression of tau was observed to cause fragmentation of βIII tubulin and a decrease in MAP2 neurites, confirming that degeneration of neurites had occurred. The antibodies used are shown below. Rabbit 4R Tau antibody (Cosmo Bio) Mouse AT-8 antibody (Thermofisher) Mouse PHF antibody (provided by Dr. Peter Davies) Rabbit βIII-tubulin antibody (Abcam) Mouse MAP2 antibody (Sigma Aldrich) Anti-Mouse IgG Alexa Fluor 555 (Thermo Fisher Scientific) Anti-Rabbit IgG Alexa Fluor 647(Thermo Fisher Scientific)

[0080] (7) Neuronal death due to overexpression of tau The decrease in cell viability due to overexpression of tau by the method described in (4) was confirmed by CellTiter Gloo assay (Promega). The results are shown in Figure 4. It was confirmed that overexpression of tau caused approximately 20% to 90% neuronal cell death in a lentivirus dose-dependent manner. Furthermore, neuronal cell death was suppressed by treatment with Tau Accell siRNA (Dharmacon, #D-001910-03), indicating that neuronal cell death occurs in a tau expression-dependent manner.

[0081] (8) Tau aggregation due to overexpression of tau Following the method described in (4), the neurons after tau overexpression were stained with a tau oligomer antibody (T22) (Millipore). The results are shown in Figure 7. Tau oligomer-positive staining was observed within the neurites, indicating that tau aggregation had occurred within the neurons. Furthermore, the length of the T22 antibody-positive neurites was quantified using Image J, confirming an increase in the length of the T22-positive neurites in the tau overexpression group compared to the non-treated group.

[0082] (9) Neuroprotective effects of tau aggregation inhibitors and microtubule stabilizers According to the method of (4), we evaluated the efficacy of methylene blue and isoproterenol, which have been reported to have tau aggregation inhibitory effects in neurons after tau overexpression, and the microtubule stabilizer epothilone D. The cytoprotective effects of the compounds were evaluated using the CellTiter Glo assay. The effect on tau aggregation was quantified by standardizing the length of Tau oligomer antibody-positive processes in neurites stained with fluorescent staining by the length of 4R Tau antibody-positive processes. The results are shown in Figure 8. Partial cell death protection effects were confirmed for methylene blue, isoproterenol, and epothilone D. Furthermore, Tau polymerization was evaluated at the compound concentrations that had cytoprotective effects. Partial Tau aggregation inhibition effects were confirmed for methylene blue and isoproterenol, which have been reported to have tau polymerization inhibitory effects, while the microtubule stabilizer epothilone D did not change Tau polymerization and showed cytoprotective effects independent of Tau polymerization.

Claims

1. A neuronal cell having only an exogenous wild-type tau gene as an introduced exogenous tau gene, the exogenous wild-type tau gene is a single tau isoform gene; Neuronal cells derived from human pluripotent stem cells derived from healthy individuals, in which expression of exogenous wild-type tau genes is condition-specifically regulated and tau aggregates when the exogenous wild-type tau gene is overexpressed.

2. The neuronal cell according to claim 1, wherein an exogenous wild-type tau gene is expressed after differentiation from a human pluripotent stem cell into a neuronal cell.

3. The neuronal cell of claim 1 or 2, wherein tau aggregation is due solely to gene expression.

4. A screening kit for a drug candidate substance that acts on changes caused by overexpression of tau, comprising the neuronal cell according to any one of claims 1 to 3.

5. A method for screening a drug candidate substance having an effect of inhibiting tau aggregation or cell death, using the neuronal cell according to any one of claims 1 to 3.

6. 4. A method for producing a neuron according to claim 1, comprising introducing an exogenous wild-type tau gene into a human pluripotent stem cell and differentiating the human pluripotent stem cell into a neuron.

7. The method according to claim 6, wherein the expression of the exogenous wild-type tau gene and the process of gene expression for differentiation of neural cells are the TET system.

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