Nerve cell analysis method, kit, and screening method for prophylactic and / or therapeutic agent for neurodegenerative disease

Abstract

The present invention addresses the problem of providing: a nerve cell analysis method with which the localized amount of TDP-43 in nerve cells can be analyzed without labeling TDP-43 with a fluorescent tag or the like and without carrying out gene transfer; a kit for carrying out the nerve cell analysis method; and a screening method for a prophylactic and / or therapeutic agent for a neurodegenerative disease. The present invention provides a nerve cell analysis method involving: a staining step for immunofluorescent staining of nerve cells using an anti-TDP-43 antibody and an antibody that recognizes a stress granule marker; a cell region identification step for identifying the cytoplasm and nuclei of the nerve cells; and an analysis step for analyzing a TDP-43-derived fluorescence signal and a stress granule marker-derived fluorescence signal in the cytoplasm, and analyzing the localized amount of TDP-43 in the nerve cells on the basis of the presence or absence of colocalization of a granular TDP-43 signal and a granular stress granule marker signal in the cytoplasm.

Description

Methods, kits, and screening methods for neuronal cell analysis and / or therapeutic agents for neurodegenerative diseases.

[0001] The present invention relates to a method for analyzing nerve cells, comprising analyzing the localization of TDP-43 in nerve cells. The present invention further relates to a kit for performing the above-described method for analyzing nerve cells. The present invention further relates to a method for screening for the prevention and / or treatment of neurodegenerative diseases using the above-described method.

[0002] Amyotrophic lateral sclerosis (ALS) is a disease characterized by progressive damage to motor nerve cells. ALS is a disease with a very poor prognosis, with patients typically dying within an average of 3 to 5 years due to respiratory failure, pneumonia, suffocation, etc. However, there is no drug that can fundamentally cure the disease, and research and development of new drugs are actively being conducted. Approximately 10% of ALS cases are familial, with disease-related gene mutations, while the remaining 90% are classified as sporadic (Non-Patent Literature 1). It is known that 95-99% of ALS patients experience aggregation of a protein called TDP-43 in the cytoplasm, and this is thought to be one of the factors causing cell damage (Non-Patent Literature 2). TDP-43 is normally localized in the cell nucleus and has a wide range of functions, including RNA splicing, localization control, and translation (Non-Patent Literature 3). For diseases involving TDP-43 aggregation, such as ALS, research is being conducted to reproduce TDP-43 aggregation in the cytoplasm in order to elucidate the pathological mechanisms and develop new therapeutic drugs. For example, there are reports of reproducing TDP-43 aggregation by forcibly expressing mutated TDP-43 (Non-Patent Literature 4). There are also reports of inducing TDP-43 aggregation by chemical stimulation; for example, there are reports of inducing aggregation in about 30 minutes by stimulation with arsenite (Non-Patent Literature 5). In addition, methods for inducing TDP-43 aggregation using oxidative stress inducers such as hydrogen peroxide (Non-Patent Literature 6), protease inhibitors such as MG-132 (Non-Patent Literature 7), and endoplasmic reticulum stress inducers such as puromycin and thapsigargin have been reported (Non-Patent Literature 8). For quantitative analysis of TDP-43 aggregation, the granular aggregation signal of TDP-43 is visualized, and the percentage of cells in which the aggregation signal can be detected, the number of aggregation signals per cell, the area, and the fluorescence intensity are evaluated (Non-Patent Literature 9). There are reports of inducing TDP-43 aggregation by forcibly expressing TDP-43 labeled with a fluorescent tag in cells, and quantitatively evaluating the degree of aggregation using the fluorescent tag (Non-Patent Literature 9). However, there are concerns that the labeling tag may affect the function and structure of the protein (Non-Patent Literature 10).While a method that induces TDP-43 aggregation and then visualizes TDP-43 by immunostaining allows for the evaluation of the protein's inherent behavior, unlike methods that use labeled TDP-43, immunostaining generally has the drawback of detecting aggregated antibodies or remnants of damaged cells as nonspecific signals (Non-Patent Documents 11 and 12).

[0003]

[0004] Richard J Mead et al., Nat Rev Drug Discov, Vol. 22, pp. 185-212, 2023. Lassi Koski et al., Int J Mol Sci, Vol. 22, Article No. 12193, 2021. Non-Nuoc Tran et al., Front Cell Dev Biol, Vol. 10, Article No. 931968, 2022. Kotaro Oiwa et al., Sci Adv, Vol. 9, Article No. 6895, 2023. Brian D Freibaum et al., J Cell Biol, Vol. 223, Article No. 202308083, 2024. Xinxin Zuo et al., Nat Struct Mol Biol, Vol. 28, pp. 132-142, 2021; Adam K. Walker et al., PLOS One, Vol. 8, paper number e81170, 2013; Mark Y. Fang et al., Neuron, Vol. 103, pp. 802-819, 2019; Chia-En Wong et al., Theranostics, Vol. 11, pp. 330-345, 2021; A. P. Araujo et al., Biochem Biophys Res Commun, Vol. 272, pp. 480-484, 2000; G. Bussolati et al., J. Clin Pathol, Vol. 61, pp. 1184-1192, 2008; Ricardo Pina et al., Int J Mol Sci, Vol. 23, Article No. 1426, 2022.

[0005] As described above, Non-Patent Literature 9 induces TDP-43 aggregation by overexpressing TDP-43 fluorescently labeled with green fluorescent protein (GFP) in cells, and quantifies the percentage of cells with aggregation signals, the area of ​​the aggregation signals, and the fluorescence intensity by detecting the GFP signal. However, since GFP-labeled TDP-43 is used, the labeling tag may be affecting the structure, localization, and function of TDP-43. Furthermore, Non-Patent Literature 9 required gene transfer.

[0006] The present invention aims to provide a method for analyzing nerve cells that can analyze the localization of TDP-43 in nerve cells without labeling TDP-43 with fluorescent tags or other means, and without gene transfer. The present invention further aims to provide a kit for performing the above-described method for analyzing nerve cells. The present invention further aims to provide a screening method for the prevention and / or treatment of neurodegenerative diseases using the above-described method.

[0007] As a result of diligent research to solve the above problems, the inventors have found that nerve cells can be analyzed by performing a staining step of immunofluorescence staining of nerve cells using an antibody, a cell region identification step of identifying the cytoplasm and nucleus of nerve cells, and an analysis step of analyzing the amount of TDP-43 localized in nerve cells based on the presence or absence of co-localization of granular TDP-43 signal and granular stress granule marker signal in the cytoplasm. The present invention was completed based on these findings.

[0008] In other words, the present invention provides the following: <1> A method for analyzing nerve cells, comprising: a staining step of immunofluorescence staining nerve cells using an anti-TDP-43 antibody and an antibody that recognizes a stress granule marker; a cell region identification step of identifying the cytoplasm and nucleus of nerve cells; and an analysis step of analyzing the fluorescence signal derived from TDP-43 and the fluorescence signal derived from a stress granule marker in the cytoplasm identified by the cell region identification step, and analyzing the amount of TDP-43 localized in the nerve cells based on the presence or absence of co-localization of the granular TDP-43 signal and the granular stress granule marker signal in the cytoplasm. <2> The analysis method according to <1>, wherein the stress granule marker is at least one selected from the group consisting of G3BP1, G3BP2, ATXN2, ATXN2L, UBAP2, UBAP2L, TIAL1, TIA1, TIAR, CARPIN1, USP10, TRIM25, TRIM56, DDX3X, FMR1, CSDE1, PRRC2C, NUFI2, and HDAC6. <3> The analysis method according to <1> or <2>, wherein the stress granule marker is at least one selected from the group consisting of G3BP1, G3BP2, ATXN2, TIAL1, and TIA1. <4> The analytical method according to any one of <1> to <3>, wherein the analytical step for analyzing the localization of TDP-43 in the nerve cells is to analyze at least one of the following indicators: (i) the percentage of cells in which granular TDP-43 signals and granular stress granule marker signals co-localize in the cytoplasm; (ii) the number of granular TDP-43 signals co-localized with stress granule markers in the cytoplasm per cell; and (iii) the area of ​​granular TDP-43 signals co-localized with stress granule marker signals in the cytoplasm per cell. <5> The analytical method according to any one of claims 1 to 4, wherein the nerve cells are nerve cells differentiated from human-derived pluripotent stem cells.<6> The analytical method according to any one of <1> to <5>, wherein the nerve cells are nerve cells in which TDP-43 aggregation is induced in the cytoplasm, obtained by culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. <7> A kit for performing the analytical method according to any one of <1> to <6>, comprising an anti-TDP-43 antibody and an antibody that recognizes a stress granule marker. <8> A screening method for the prevention and / or treatment of neurodegenerative diseases, comprising: a test substance contact step of contacting nerve cells with a test substance; an analysis step of measuring the amount of TDP-43 localized in the cytoplasm of the nerve cells after the test substance contact step by the analytical method according to any one of <1> to <6>; and a selection step of selecting the test substance as a candidate for the prevention and / or treatment of neurodegenerative diseases based on the amount of TDP-43 localized in the analysis step. <9> The screening method according to <8>, wherein, in the selection step described above, cells in which the localization of TDP-43 in the cytoplasm of the nerve cells is reduced compared to the case in which the test substance is not brought into contact are selected as candidates for the prevention and / or treatment of neurodegenerative diseases. <10> The screening method according to <8> or <9>, wherein the nerve cells are nerve cells differentiated from human-derived pluripotent stem cells. <11> The screening method according to any one of <8> to <10>, wherein the nerve cells are nerve cells in which TDP-43 aggregation is induced in the cytoplasm, obtained by culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death.<12> The above neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), familial frontotemporal dementia-parkinsonism linked to chromosome 17 (FTDP-17), Paget's disease of bone and inclusion body myopathy with frontotemporal dementia (IBMPFD), Perry syndrome, Parkinson's disease (PD), Parkinson's dementia complex (PDC), familial British dementia (FBD), Huntington's disease (HD), and Machado Joseph disease. A screening method according to any one of <8> to <11>, wherein the selected disease is at least one from the group consisting of (MJD / SCA3), Alzheimer's disease (AD), Lewy body dementia (DLB), corticobasal degeneration (CBD), argyrophilic granule disease / argyrophilic granule dementia (AGD), diffuse neurofibrillary tangle disease with calcification (DNTC), and progressive supranuclear palsy (PSP).

[0009] This invention provides a method for quantitatively analyzing the degree of TDP-43 aggregation using immunostaining after TDP-43 aggregation has formed, utilizing a signal in which TDP-43 and stress granule markers co-localize. Unlike methods that visualize TDP-43 using fluorescently labeled fusion proteins, this invention allows for the evaluation of the intrinsic effect of TDP-43 aggregation. Furthermore, since this invention does not require gene transfer, quantitative evaluation can be performed simply. Moreover, by limiting the target of quantitative evaluation to the TDP-43 signal co-localized with the stress granule signal, the influence of nonspecific signals, which is a challenge in immunostaining, can be significantly suppressed.

[0010] Figure 1 shows images of unstimulated nerve cells (TDP-43, G3BP1, Hoechst33342). Figure 2 shows images of stimulated nerve cells (TDP-43, G3BP1, Hoechst33342). Figure 3 shows the percentage of cells positive for granular TDP-43 signaling in the cytoplasm. Figure 4 shows the percentage of cells co-localizing granular TDP-43 and G3BP1 signaling in the cytoplasm. Figure 5 shows the number of granular TDP-43 signals per cell in the cytoplasm. Figure 6 shows the number of granular TDP-43 signals co-localized with G3BP1 signaling per cell in the cytoplasm. Figure 7 shows the area per cell of granular TDP-43 signaling in the cytoplasm. Figure 8 shows the area per cell of granular TDP-43 signals co-localized with G3BP1 signals in the cytoplasm. Figure 9 shows the results of quantitative evaluation of TDP-43 aggregation in the cytoplasm using only the TDP-43 signal or using the TDP-43 and G3BP2 signals. Figure 10 shows the results of quantitative evaluation of TDP-43 aggregation in the cytoplasm using only the TDP-43 signal or using the TDP-43 and ATXN2 signals. Figure 11 shows the results of quantitative evaluation of TDP-43 aggregation in the cytoplasm using only the TDP-43 signal or using the TDP-43 and TIAL1 signals. Figure 12 shows the results of quantitative evaluation of TDP-43 aggregation in the cytoplasm using only the TDP-43 signal or using the TDP-43 and TIA1 signals.

[0011] The embodiments of the present invention will be described in detail below.

[0012] <Method for Analyzing Nerve Cells> The method for analyzing nerve cells of the present invention includes: a staining step of immunofluorescence staining nerve cells using an anti-TDP-43 antibody and an antibody that recognizes a stress granule marker; a cell region identification step of identifying the cytoplasm and nucleus of nerve cells; and an analysis step of analyzing the fluorescence signals derived from TDP-43 and stress granule markers in the cytoplasm identified by the cell region identification step, and analyzing the amount of TDP-43 localized in the nerve cells based on the presence or absence of co-localization of granular TDP-43 signals and granular stress granule marker signals in the cytoplasm.

[0013] In this invention, the degree of TDP-43 aggregation is analyzed using a signal in which TDP-43 and a stress granule marker colocalize by immunohistochemistry. According to this invention, TDP-43 aggregation signals related to disease states can be detected with high accuracy. This invention can be used for the development of new therapeutic drugs, elucidation of disease mechanisms, and biomarker discovery for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), in which TDP-43 aggregation is known to be a characteristic feature of the disease state.

[0014] The nerve cells are not particularly limited, but are preferably motor nerve cells, cerebral cortical excitatory nerve cells, or substantia nigra nerve cells, more preferably motor nerve cells or cerebral cortical excitatory nerve cells, and even more preferably motor nerve cells.

[0015] While not particularly limited, nerve cells differentiated from human-derived pluripotent stem cells may be used. For example, nerve cells obtained by culturing human-derived pluripotent stem cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death, and in which TDP-43 aggregation is induced in the cytoplasm, may be used. Culturing nerve cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death will also be referred to as "Step A" below.

[0016] Examples of human-derived pluripotent stem cells include pluripotent stem cells derived from specimens without mutations in disease-related genes that cause nervous system diseases, pluripotent stem cells derived from specimens collected from healthy humans (healthy individuals) without nervous system diseases, pluripotent stem cells derived from specimens collected from patients with diseases, or pluripotent stem cells derived from specimens with mutations in disease-related genes. "Without mutations in disease-related genes" means not having mutations in disease-related genes that cause nervous system diseases. That is, even if there are mutations in the gene, if they are mutations that do not cause the disease, they shall be interpreted as not having mutations in disease-related genes. Also, in the case of "having mutations in disease-related genes", the mutations may be endogenous gene mutations originally possessed by the patient or exogenous gene mutations with artificially introduced gene mutations. By producing nerve cells in which TDP-43 aggregation is induced in the cytoplasm using pluripotent stem cells derived from specimens collected from patients with diseases or pluripotent stem cells derived from specimens with mutations in disease-related genes, it is assumed that the pathological conditions of diseases caused by specimen-derived diseases or mutations in disease-related genes can be mimicked.

[0017] Examples of human-derived pluripotent stem cells include human iPS cells (human induced pluripotent stem cells), human ES cells (human embryonic stem cells), human mesenchymal stem cells, and the like. Human iPS cells are preferred but not particularly limited. Human iPS cells are iPS cells produced from human cells.

[0018] ES cells can be established, for example, by culturing early embryos before implantation, the inner cell mass that makes up the aforementioned early embryo, or single blastomeres (Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Thomson, JA et al., Science, 282, 1145-1147 (1998)). Early embryos may be produced by nuclear transfer of somatic cell nuclei (Wilmut et al. (Nature, 385, 810 (1997)), Cibelli et al. (Science, 280, 1256 (1998)), 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. Parthenogenesis embryos may also be used as early embryos (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 paper, regarding the production of ES cells, 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, P.M. et al. Methods in Enzymology, Vol. 365, 2003, etc. are also helpful. Note that fusion ES cells obtained by cell fusion of 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 preservation institutions or are commercially available. For example, for human ES cells, they are available from the Institute for Frontier Medical Sciences, Kyoto University (such as KhES-1, KhES-2 and KhES-3), WiCell Research Institute, ESI BIO, etc.

[0020] iPS cells refer to cells having pluripotency (multipotency) and proliferation ability, which are produced by reprogramming somatic cells by introducing reprogramming factors. iPS cells show properties similar to ES cells. The somatic cells used for the production of iPS cells are not particularly limited, and may be differentiated somatic cells or undifferentiated stem cells. iPS cells can be produced by known methods, etc. It is also naturally assumed that iPS cell production methods to be developed in the future will be applied.

[0021] The most basic method for generating iPS cells involves introducing 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). For human iPS cells, there are reports of establishment by introducing four factors: Oct3 / 4, Sox2, Lin28, and Nanog (Yu J, et al: Science 318 (5858), 1917-1920, 2007). The establishment of iPS cells by introducing three factors excluding 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 Oct3 / 4 only (Kim JB, et al: Cell 136 (3), 411-419, 2009) has also been reported. Furthermore, methods for introducing proteins, which are the expression products of genes, into cells have 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, there are reports that the efficiency of synthesis and the number of introduced factors can be improved by using inhibitors against histone methyltransferase G9a such as BIX-01294, or histone deacetylase inhibitors such as valproic acid (VPA) or BayK8644 (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).Gene transfer methods are also being investigated, including 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), and transposon vectors (Woltjen K, Michael IP, Mohseni P, et al: Nature 458, 766-770, 2009; Kaji K, Norrby K, Paca A, et al: Nature 458, 771-775, 2009; Yusa K, Rad R, Takeda J, et al: Nat.Methods 6, 363-369). Techniques have been developed that utilize episomal vectors (Yu J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009) for gene transfer.

[0022] Cells that have undergone transformation into iPS cells, i.e., reprogramming, can be selected based on indicators such as the expression of pluripotent stem cell markers (undifferentiated markers) including Fbxo15, Nanog, Oct3 / 4, Fgf-4, Esg-1, and Cript. The selected cells can then be harvested as iPS cells.

[0023] In addition to the method of producing iPS cells through direct reprogramming via gene expression, it is also possible to induce iPS cells from somatic cells by adding compounds (Hou P et al: Science 341 (6146), 651-654, 2013).

[0024] iPS cells can also be obtained from sources such as FUJIFILM Cellular Dynamics, Inc. (FCDI), Kyoto University, or the RIKEN BioResource Research Center.

[0025] Methods for obtaining nerve cells differentiated from human-derived pluripotent stem cells include, for example, inducing them from somatic cells collected from healthy individuals (healthy subjects) without disease-related gene mutations or neurological diseases, or from somatic cells collected from patients with neurological diseases, or from established human iPS cell lines.

[0026] There are no particular limitations on the methods for inducing differentiation of human-derived pluripotent stem cells into nerve cells, but these include methods that involve generating neural stem cells from pluripotent stem cells using small molecule compound treatment and then inducing them into nerve cells, and methods that involve directly inducing them into nerve cells through gene expression, etc.

[0027] Methods for inducing differentiation of pluripotent stem cells into nerve cells include, for example: (1) culturing in serum-free medium to form embryoid bodies (cell aggregates containing neural progenitor cells) and then differentiating them (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) culturing on stromal cells and then differentiating them (SDIA method: Kawasaki H., et al, Neuron, 28: 31-40, 2000); (3) culturing on Matrigel with added drugs and then differentiating them (Chambers SM, et al, Nat. Biotechnol., 27: 275-280, (2009); (4) A method of differentiation by culturing in a medium containing small molecule compounds as cytokine substitutes (U.S. Patent No. 5,843,780); (5) A method of differentiation by introducing and expressing neuroinducible factors (such as the gene encoding neurogenin2 (NGN2) protein) into pluripotent stem cells (WO2014 / 148646; and Zhang Y., et al, Neuron, 78: 785-98, 2013); (6) A method of differentiation by introducing and expressing miR-9 / 9*-124 into pluripotent stem cells; and combinations of these methods.

[0028] Of the above, (5) the method of introducing a gene encoding the NGN2 protein into pluripotent stem cells and expressing it is preferred because it can produce mature nerve cells in a short period of time and with high efficiency.

[0029] Neurogenein 2 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. The neurogenein 2 gene (Official full name: neurogenein 2, Official symbol: NEUROG2, also called the Ngn2 gene) is the DNA that encodes the neurogenein 2 protein, and examples include DNA having the nucleotide sequences of NM_009718 (mouse) or NM_024019 (human), which are registered as standard sequences, or their transcription variants. Alternatively, it may be DNA having sufficient complementarity to hybridize under stringent conditions to nucleic acids having the above standard sequences and transcription variants.

[0030] As excitatory neurons of the cerebral cortex differentiated from human iPS cells, it is preferable to use cells produced by forcibly expressing the Ngn2 gene from human iPS cells.

[0031] As nerve cells differentiated from human-derived pluripotent stem cells, commercially available nerve cells may be used, for example, iCell® motor nerve cells (FCDI, C1050, C1048).

[0032] As substantia nigra neurons differentiated from human-derived pluripotent stem cells, commercially available neurons may be used, for example, iCell® dopamine neurons (FCDI, C1087, C1028).

[0033] A nerve cell preferably expresses at least one marker gene specific to nerve cells, which consists of β-III tubulin, NeuN, N-CAM (neural cell adhesion molecule), and MAP2 (microtubule-associated protein 2), and is a cell having β-III tubulin-positive protrusions (hereinafter referred to as neurites).

[0034] The expression level of the marker gene can usually be analyzed by the production amount of the transcript corresponding to the gene, or the production amount, activity, etc. of its translation product. The measurement of the expression level can be performed by measuring the mRNA which is the transcript of the gene or the protein which is the translation product of the gene, but it is preferably performed by measuring the mRNA or its reverse transcription product cDNA. The detection or measurement of the expression of the translation product (protein) can be performed by immunocytochemical staining using an antibody to detect the protein in the cell.

[0035] Step A can be performed by seeding cells in a medium and starting the culture. The lower limit value of the cell density at the time of seeding the cells is not particularly limited. For example, it is preferably 1×10 ４ cells / cm ２ or more, more preferably 3×10 ４ cells / cm ２ or more, even more preferably 6×10 ４ cells / cm [[ID=,17]] ２ or more, still more preferably 12×10 ４ cells / cm ２ or more. The upper limit value of the cell density is not particularly limited. For example, it may be 50×10 ４ cells / cm ２ or less, preferably less than 40×10 ４ cells / cm ２ more preferably less than 30×10 ４ cells / cm ２ even more preferably less than 20×10 ４ cells / cm ２ still more preferably. The cell density is preferably 1×10 ４ cells / cm ２ or more and 50×10４ cells / cm ２ The following, more preferably 3 × 10 ４ cells / cm ２ 40 x 10 ４ cells / cm ２ The following, and more preferably 6 × 10 ４ cells / cm ２ 30 x 10 ４ cells / cm ２ The following, and more preferably 12 × 10 ４ cells / cm ２ The above 20 x 10 ４ cells / cm ２ The following applies:

[0036] In step A, nerve cells differentiated from human-derived pluripotent stem cells are cultured in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death. As a concentration that does not induce cell death, for example, if the number of viable cells is 20% or more compared to the unstimulated condition, it can be determined that strong cell death has not been induced, and it is preferable to use a concentration that can reproduce such conditions.

[0037] Examples of protein synthesis inhibitors include at least one selected from the group consisting of aminonucleoside antibiotics, peptidyl nucleoside antibiotics, aminoglycoside antibiotics, and glutarimide antibiotics. Among these, aminonucleoside antibiotics and peptidyl nucleoside antibiotics are preferred.

[0038] Examples of aminonucleoside antibiotics include puromycin. Examples of peptidyl nucleoside antibiotics include blasticidin S, nikkomycin Z, and polyoxin D. Examples of aminoglycoside antibiotics include G-418, hydroxycin B, kanamycin, gentamycin, and streptomycin. Examples of glutarimide antibiotics include cycloheximide and actiketal.

[0039] When the protein synthesis inhibitor is puromycin, the concentration that does not induce cell death is preferably greater than 5 μg / mL to 400 μg / mL, more preferably greater than 5 μg / mL to 100 μg / mL, even more preferably greater than 5 μg / mL to 40 μg / mL, and even more preferably between 10 μg / mL and 15 μg / mL. A concentration of 400 μg / mL or less is preferable from the viewpoint of cytotoxicity.

[0040] When the protein synthesis inhibitor is Blasticidin S, the concentration that does not induce cell death is preferably greater than 1 μg / mL to 400 μg / mL, more preferably greater than 1 μg / mL to 90 μg / mL, even more preferably greater than 1 μg / mL to 40 μg / mL, and even more preferably between 10 μg / mL and 40 μg / mL. A concentration of 400 μg / mL or less is preferable from the viewpoint of cytotoxicity.

[0041] When the protein synthesis inhibitor is G-418, the concentration that does not induce cell death is preferably greater than 600 μg / mL to 20 mg / mL, more preferably greater than 600 μg / mL to 2000 μg / mL, and even more preferably between 1500 μg / mL and 2000 μg / mL. A concentration of 20 mg / mL or less is preferable from the viewpoint of cytotoxicity.

[0042] When the protein synthesis inhibitor is hydroxycin B, the concentration that does not induce cell death is preferably greater than 300 μg / mL to 20 mg / mL, more preferably greater than 300 μg / mL to 2000 μg / mL, and even more preferably between 600 μg / mL and 1500 μg / mL. A concentration of 20 mg / mL or less is preferable from the viewpoint of cytotoxicity.

[0043] When the protein synthesis inhibitor is cycloheximide, the concentration that does not induce cell death is preferably greater than 10 μmol / L to 400 μmol / L, more preferably greater than 10 μmol / L to 40 μmol / L, and even more preferably between 15 μmol / L and 20 μmol / L. A concentration of 400 μmol / L or less is preferable from the viewpoint of cytotoxicity.

[0044] The culture time in step A (i.e., the culture time in the culture medium containing the protein synthesis inhibitor) is preferably 2 to 72 hours, more preferably 6 to 60 hours, even more preferably 12 to 50 hours, and particularly preferably 24 to 48 hours. From the viewpoint of cytotoxicity, a culture period of 72 hours or less in step A is preferable.

[0045] The culture medium can be selected from known media or commercially available media. The culture medium used for cultivation can be prepared by adding additives to the basal medium. Here, the basal media are, for example, DMEM, DMEM (Dulbecc's Modified Eagle Medium) / F12, BrainPhys® Neuronal Medium, Neurobasal® Medium-A, Neurobasal® Medium, Neuroal Progenitor Basal Medium, NS-A Basal Medium, Basal Medium Eagle (BME), BGJb Medium, CMRL 1066 Medium, Glasgow Minimum Essential Medium (MEM), Improved MEM Zinc Option, and Iscove's Examples include Modified Dulbecco's Medium (IMDM), Medium 199, Eagle MEM, αMEM, Ham's F12 Medium, RPMI 1640 Medium, and Fischer's Medium. Furthermore, a single culture medium may be used, or a combination of two or more culture media may be employed.

[0046] As for the culture medium, Neurobasal ＴＭ Medium, Neurobasal ＴＭ -A Medium, Neurobasal ＴＭ Plus Medium, DMEM / F12, and DMEM are preferred, and Neurobasal ＴＭ Medium is particularly preferred.

[0047] Additives may be added if they do not exhibit substantially antioxidant activity, or if added at concentrations that do not exhibit substantially antioxidant activity. Specifically, examples include, but are not limited to, serum, retinoic acid, Wnt, BMP, bFGF, EGF, HGF, Sonic hedgehog (Shh), interleukins, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B-27® supplement (antioxidant-free), and ITS-supplement. A preferred additive is B-27® supplement (antioxidant-free).

[0048] In one embodiment of the present invention, step A may include step A1, in which hydrogen peroxide is added to the culture medium at a concentration of 0.5 mmol / L to 2.0 mmol / L (preferably 0.8 mmol / L to 1.5 mmol / L), followed by cultivation. The cultivation time for step A1 is preferably 30 to 120 minutes, more preferably 30 to 90 minutes, and even more preferably 30 to 60 minutes or 45 to 90 minutes.

[0049] For culturing nerve cells, you can simply select general cell culture conditions: 37°C, 5% CO2. ２ Examples of conditions include the following. During cultivation, the culture medium may be changed at appropriate intervals (preferably once every 1 to 7 days, more preferably once every 2 to 3 days), but it is preferable not to change the culture medium during the cultivation period.

[0050] Nerve cells are preferably cultured in two dimensions. Cell culture vessels such as plates, dishes, cell culture inserts, and cell culture flasks can be used for cell culture.

[0051] As described above, nerve cells in which TDP-43 aggregation is induced in the cytoplasm can be produced.

[0052] (Staining process) The staining process involves immunofluorescence staining of nerve cells using an anti-TDP-43 antibody and an antibody that recognizes stress granule markers.

[0053] TDP-43, also known as TAR DNA-binding protein of 43 kDa, is a nuclear-localized RNA-binding protein. TDP-43 is involved in the transcription and splicing of various genes. The stress granule marker is preferably a stress granule protein marker. As a stress granule marker, at least one selected from the group consisting of G3BP1, G3BP2, ATXN2, ATXN2L, UBAP2, UBAP2L, TIAL1, TIA1, TIAR, CARPIN1, USP10, TRIM25, TRIM56, DDX3X, FMR1, CSDE1, PRRC2C, NUFI2, and HDAC6 can be used. The stress granule marker can be at least one selected from the group consisting of G3BP1, G3BP2, ATXN2, TIAL1, and TIA1, which is particularly preferable.

[0054] Any antibody capable of binding to TDP-43 is acceptable as the anti-TDP-43 antibody. Any antibody capable of binding to stress granule markers is acceptable as the antibody that recognizes stress granule markers. The anti-TDP-43 antibody and the antibody that recognizes stress granule markers may be commercially available or prepared appropriately by conventional methods, and may be monoclonal or polyclonal antibodies. The anti-TDP-43 antibody and the antibody that recognizes stress granule markers can be prepared by methods utilizing cell fusion technology or genetic recombination technology.

[0055] Antibodies that recognize anti-TDP-43 antibodies and stress granule markers are Fab, Fab', and F(ab') of the antibody. ２ Fv, Fd, single-stranded Fv (scFv), disulfide-bonded Fv (sdFv), V Ｌ , V Ｈ , Diamond Body ((V Ｌ -V Ｈ ) ２ Or (V Ｈ -V Ｌ ) ２ ), Triabody (trivalent antibody), Tetrabody (tetravalent antibody), Minibody ((scF Ｖ -C Ｈ 3)２ ), IgG-delta-CH2, scFv-Fc, (scFv) ２ -Fc fragments, etc., may also be used.

[0056] Examples of commercially available anti-TDP-43 antibodies include the anti-TDP-43 antibody (Proteintech, 10782-2-AP). Examples of commercially available antibodies that recognize stress granule markers include the anti-G3BP1 antibody (Proteintech, 66486-1-Ig).

[0057] Specific examples of immunofluorescence staining include the following method: As primary antibodies, an anti-TDP-43 antibody and an antibody that recognizes stress granule markers are used. As secondary antibodies, a labeled secondary antibody labeled with a first fluorescent substance that can detect the above-mentioned anti-TDP-43 antibody and a labeled secondary antibody labeled with a second fluorescent substance that can detect the antibody that recognizes the above-mentioned stress granule markers are used. Different fluorescent substances are used for the first and second fluorescent substances.

[0058] As fluorescent substances, you can use HiLyte dyes, fluorescein, dansyl, fluorescein camine, coumarin, naphthylamine, fluorescein isothiocyanate (FITC), rhodamine, rhodamine X isothiocyanate, sulforodamine 101, Lucifer Yellow, acridine, acridine isothiocyanate, riboflavin, or derivatives thereof. Specific examples of fluorescent substances include HiLyte dyes such as HiLyte Fluor 647, HiLyte Fluor 488, HiLyte Fluor 555, HiLyte Fluor 680, and HiLyte Fluor 750 (all trade names of HiLyte Bioscience, Inc.), and Alexa dyes such as Alexa Fluor Dye 350, Alexa Fluor Dye 430, Alexa Fluor Dye 488, Alexa Fluor Dye 532, Alexa Fluor Dye 546, Alexa Fluor Dye 555, Alexa Fluor Dye 568, Alexa Fluor Dye 594, Alexa Fluor Dye 633, Alexa Fluor Dye 647, Alexa Fluor Dye 660, Alexa Fluor Dye 680, Alexa Fluor Dye 700, and Alexa Fluor Dye 750 (all trade names of Molecular Probes). CyDye dyes such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, etc. (all trade names of Amersham Biosciences) can be used.

[0059] Nerve cells are reacted with a primary antibody and a labeled secondary antibody to generate a complex of TDP-43, the primary antibody, and the labeled secondary antibody, as well as a complex of a stress granule marker, the primary antibody, and the labeled secondary antibody.

[0060] (Cell Region Identification Step) The cell region identification step is a step in which the cytoplasm and nucleus of nerve cells are identified. Identifying the cytoplasm and nucleus of nerve cells can be done, for example, by staining the nucleus with a reagent that can stain the cell nucleus. As a reagent that can stain the cell nucleus, Hoechst 33342 (Dojindo, 346-07951) can be used.

[0061] In one example of the present invention, after performing immunofluorescence staining and cell nucleus staining as described above, images of the cells can be obtained. Images of the cells can be obtained, for example, by photographing the cells using a confocal quantitative image cytometer.

[0062] (Analysis Process) The analysis process involves analyzing the fluorescent signals derived from TDP-43 and stress granule markers in the cytoplasm identified by the cell region identification process, and analyzing the amount of TDP-43 localized in nerve cells based on the presence or absence of co-localization of the granular TDP-43 signal and the granular stress granule marker signal in the cytoplasm.

[0063] The localization of TDP-43 in nerve cells can be analyzed using image analysis software on a confocal quantitative image cytometer (e.g., Yokogawa Electric Corporation, CellVoyager CQ1) to recognize granular signals of TDP-43 and stress granule markers (e.g., G3BP1) and evaluate the degree of aggregation.

[0064] The granular signals of TDP-43 or stress granule markers (e.g., G3BP1) refer to, for example, fluorescent signals derived from TDP-43 or stress granule markers in the cytoplasm identified by the cell region recognition step, which are aggregated and have a certain size. The size of the granular signals is not particularly limited, but for example, 0.1 to 100 μm. ２ Signals within this range can be counted as granular signals.

[0065] When quantifying the percentage of cells with granular TDP-43 signals, nonspecific signals are often detected, making it difficult to accurately analyze the localization of TDP-43 in nerve cells. In this invention, by quantifying the percentage of cells with granular TDP-43 signals co-localized with granular stress granule marker signals, it becomes possible to accurately detect TDP-43 aggregation signals related to pathological conditions.

[0066] The analysis step for analyzing the localization of TDP-43 in nerve cells preferably involves analyzing at least one of the following indicators: (i) the percentage of cells in which granular TDP-43 signals and granular stress granule marker signals co-localize in the cytoplasm; (ii) the number of granular TDP-43 signals co-localized with stress granule markers in the cytoplasm per cell; and (iii) the area of ​​granular TDP-43 signals co-localized with stress granule marker signals in the cytoplasm per cell.

[0067] <Kit> The present invention further relates to a kit for performing a method for analyzing nerve cells according to the present invention, comprising an anti-TDP-43 antibody and an antibody that recognizes stress granule markers.

[0068] The anti-TDP-43 antibody and the antibody that recognizes the stress granule marker can be those described above in this specification. The anti-TDP-43 antibody and the antibody that recognizes the stress granule marker may be in solution form, such as a suspension in a suitable buffer, or they may be frozen or lyophilized.

[0069] <Screening method for the prevention and / or treatment of neurodegenerative diseases> The nerve cell analysis method according to the present invention allows for the analysis of the localization amount of TDP-43 in nerve cells. Therefore, the nerve cell analysis method according to the present invention can be used for screening new drugs useful for neurodegenerative diseases in which TDP-43 aggregation is known to play a significant role in the onset and progression of the disease.

[0070] The present invention provides a screening method for neurodegenerative diseases, comprising: a test substance contact step of contacting nerve cells with a test substance; an analysis step of measuring the amount of TDP-43 localized in the cytoplasm of the nerve cells after the test substance contact step using an analysis method according to the present invention; and a selection step of selecting the test substance as a candidate for the prevention and / or treatment of neurodegenerative diseases based on the amount of TDP-43 localized in the analysis step.

[0071] In one embodiment of the screening method of the present invention, in the selection step, nerve cells in which the localization of TDP-43 in the cytoplasm of the nerve cells is reduced compared to when the test substance is not in contact with the nerve cells may be selected as candidates for the prevention and / or treatment of neurodegenerative diseases.

[0072] In one embodiment of the screening method of the present invention, nerve cells may be those differentiated from human-derived pluripotent stem cells. In one embodiment of the screening method of the present invention, nerve cells may be those obtained by culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death, thereby in which TDP-43 aggregation is induced in the cytoplasm. Details of the nerve cells described above are as stated herein.

[0073] 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 small molecule compounds, natural compounds, cell extracts, extracellular vesicles, plant extracts, animal tissue extracts, plasma, extracts derived from marine organisms, cell culture supernatants, and microbial fermentation products.

[0074] Furthermore, test substances can be obtained using any of the many combinatorial library methods known in the art, including (1) the biological library method, (2) the synthetic library method using deconvolution, (3) the one-bead-one-compound library method, and (4) the synthetic library method using affinity chromatography sorting. The biological library method using affinity chromatography sorting is limited to peptide libraries, but the 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 molecular library synthesis methods 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. (1994) J.Med.Chem. 37: 1233-51).Compound libraries can be prepared in solution (see Houghten (1992) Bio / Techniques 13: 412-21), beads (Lam (1991) Nature 354: 82-4), tips (Fodor (1993) Nature 364: 555-6), bacteria (US Patent No. 5,223,409), spores (US Patent 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. (1990)). It can be manufactured as Proc.Natl.Acad.Sci.USA 87: 6378-82; Felici (1991) J.Mol.Biol. 222: 301-10; U.S. Patent Application Publication No. 2002 / 0103360).

[0075] The process of culturing nerve cells in the presence of the test substance may be carried out by adding the test substance to the nerve cell culture medium. The culture time is not particularly limited as long as a change in the indicator can be confirmed, but for example, it may be 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 added test substance can be adjusted as appropriate depending on the type of compound (solubility, toxicity, etc.).

[0076] The culture medium used when culturing nerve cells in the presence of a test substance is not particularly limited, as long as it is a culture medium capable of culturing nerve cells.

[0077] The culture temperature when culturing nerve cells in the presence of the test substance is not particularly limited, but is about 30 to 40°C, preferably about 37°C, CO ２ The culture is carried out in an atmosphere containing CO ２ The concentration is preferably about 2-5%.

[0078] Neurodegenerative diseases include, but are not limited to, at least one selected from the group consisting of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), familial frontotemporal dementia-parkinsonism linked to chromosome 17 (FTDP-17), Paget's disease of bone and inclusion body myopathy with frontotemporal dementia (IBMPFD), Perry syndrome, Parkinson's disease (PD), Parkinson's dementia complex (PDC), familial British dementia (FBD), Huntington's disease (HD), Machado Joseph disease (MJD / SCA3), Alzheimer's disease (AD), Lewy body dementia (DLB), corticobasal degeneration (CBD), argyrophilic grain disease / argyrophilic grain dementia (AGD), diffuse neurofibrillary tangle disease with calcification (DNTC), and progressive supranuclear palsy (PSP).

[0079] Preferred neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Huntington's disease (HD), or Alzheimer's disease (AD), with amyotrophic lateral sclerosis (ALS) being more preferred.

[0080] The present invention will be specifically described by the following examples, but the present invention is not limited to the scope of these examples.

[0081] Test Example 1: Reproduction of TDP-43 aggregation by various protein synthesis inhibitors <Plate coating> iMatrix-511 silk (Matrixome, 892021) was diluted 166.7 times with PBS and added at a rate of 70 μL / well to a PDL-coated 96-well plate (Corning, 356640), and incubated at 37°C for 1 day.

[0082] <Cell Seeding> The following culture medium was prepared. Frozen stocks of nerve cells, produced by forced expression of the Ngn2 gene in iPS cells, were thawed in a 37°C warm bath. After thawing, the cells were added to the culture medium and centrifuged at 600 × g at room temperature for 5 minutes. After centrifugation, the supernatant was removed, the cells were suspended in 1 mL of culture medium, and the number of cells was counted. Then, the cells were diluted with culture medium and rehydrated in 100 μL / well (5 × 10⁻⁶). ４ Seeds were sown in cells / well, at 37°C and 5% CO2. ２ The cells were cultured under specific conditions.

[0083]

[0084] <Induction of TDP-43 aggregation and cell fixation> Three days after culturing nerve cells, puromycin was added to the culture medium to a concentration of 10 μg / mL. After 23 hours, hydrogen peroxide was added to a final concentration of 1 mM. One hour after hydrogen peroxide treatment, the cells were fixed by treating them for 30 minutes with formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555).

[0085] <Immunofluorescence Staining> After fixing the cells, they were washed with PBS(-) and then blocked and permeabilized by treating them for 30 minutes with 1% and 0.2% solutions of BSA (Sigma Aldrich, A2153) and Triton X-100 solution (BioVision, 2104-100) diluted with PBS(-) (1% BSA / 0.2% Triton-X). Subsequently, as a primary antibody reaction, the cells were treated with 1000-fold diluted anti-TDP-43 antibody (Proteintech, 10782-2-AP) and 1000-fold diluted anti-G3BP1 antibody (Proteintech, 66486-1-Ig) in 1% BSA / 0.2% Triton-X, and left to stand overnight at 4°C.

[0086] The following day, after washing with PBS(-), the samples were treated with 1000-fold diluted Goat anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, A11008), 1000-fold diluted Goat anti-rabbit Alexa Fluor 594 (Thermo Fisher Scientific, A11005), and 1000-fold diluted Hoechst 33342 (Dojindo, 346-07951) as secondary antibody reactions, and left to stand at room temperature for 60 minutes. After washing with PBS(-), images were acquired using a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1).

[0087] <Evaluation of TDP-43 Aggregation> Using image analysis software from a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and G3BP1 were recognized, and the degree of aggregation was evaluated. In addition, the nuclear region was recognized using the signal of Hoechst 33342, and the portion of the TDP-43 signal region excluding the nuclear region was recognized as cytoplasm. In unstimulated neurons, granular TDP-43 signals in the cytoplasm were hardly observed (Figure 1, left center, bottom left). Also, no strong signal of G3BP1 was observed (Figure 1, center top). On the other hand, in neurons stimulated with puromycin and hydrogen peroxide, many granular TDP-43 signals were observed extranuclear (Figure 2, bottom left white arrow). Also, many granular G3BP1 signals were observed extranuclear (Figure 2, center bottom white arrow). Furthermore, numerous granular signals of TDP-43 co-localized with G3BP1 outside the nucleus were observed (white arrow in the lower right of Figure 2). It was confirmed that the amount of granular signals of TDP-43 differed significantly depending on whether stimulation was present or not.

[0088] However, when the proportion of cells with granular TDP-43 signals was quantified, many nonspecific signals were detected, and little difference was observed between the presence or absence of stimulation (Figure 3).

[0089] On the other hand, when the percentage of cells with granular TDP-43 signals co-localized with G3BP1 signals was quantified, a clear difference between cells with and without stimulation was observed, reflecting the results of immunohistochemistry (Figure 4). In other words, TDP-43 aggregation signals related to the disease state could be detected with high accuracy.

[0090] Regarding the number of TDP-43 signals per cell (Figures 5 and 6) and area (Figures 7 and 8), by evaluating TDP-43 co-localized with G3BP1, rather than TDP-43 alone, we were able to significantly reduce nonspecific quantitative values ​​without stimulation. In summary, by quantitatively analyzing TDP-43 aggregation using signals co-localized with stress granule markers, we were able to suppress the detection of nonspecific signals and accurately detect the target TDP-43 aggregation signal.

[0091] Test Example 2: Quantitative determination of TDP-43 aggregation using various stress granule markers

[0092] Plate coating and cell seeding were performed using the same method as in Experimental Example 1.

[0093] <Induction of TDP-43 aggregation and cell fixation> Three days after culturing nerve cells, puromycin was added to the culture medium to a concentration of 10-20 μg / mL. 16-24 hours after treatment, cells were fixed by treating them for 30 minutes with a formaldehyde solution (Fujifilm Wako, 061-00416) diluted 10-fold with PBS(-) (Fujifilm Wako, 166-23555).

[0094] <Fluorescent Immunostaining> Fluorescent immunostaining was performed in the same manner as in Test Example 1, except for the primary antibody. The following antibodies were used as primary antibodies, diluted 1000 times: • TDP-43 antibody (Proteintech, 10782-2-AP) • TDP-43 antibody (Proteintech, 67345-1-Ig) • G3BP2 antibody (Abclonal, A6026) • ATXN2 antibody (Abclonal, A16666) • TIAL1 antibody (Abclonal, A6075) • TIA1 antibody (Thermo Fisher Scientific, MA5-26474)

[0095] <Evaluation of TDP-43 Aggregation> After immunofluorescence staining, stained cells were imaged using a confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1) and images were acquired. Using the image analysis software of the confocal quantitative image cytometer (Yokogawa Electric Corporation, CellVoyager CQ1), granular signals of TDP-43 and various stress granule markers were recognized, and the degree of TDP-43 aggregation was evaluated. G3BP2, ATXN2, TIAL1, and TIA1 were evaluated as stress granule markers. As a result, when TDP-43 aggregation was quantified using a granular signal of TDP-43 alone, many nonspecific signals were detected in Vehicle, and the difference between the presence and absence of stimulation was small (Figures 9-12 (a)-(c)). However, when quantified using a granular signal of TDP-43 co-localized with stress granule markers, the difference between the presence and absence of stimulation was clearly confirmed (Figures 9-12 (d)-(f)). Therefore, by quantitatively analyzing TDP-43 aggregation using a signal in which TDP-43 and various stress granule markers co-localize, the detection of nonspecific signals was suppressed, and the target TDP-43 aggregation signal was detected with high accuracy.

Claims

1. A method for analyzing nerve cells, comprising: a staining step of immunofluorescence staining nerve cells using an anti-TDP-43 antibody and an antibody that recognizes a stress granule marker; a cell region identification step of identifying the cytoplasm and nucleus of nerve cells; and an analysis step of analyzing the fluorescence signals derived from TDP-43 and stress granule markers in the cytoplasm identified by the cell region identification step, and analyzing the amount of TDP-43 localized in the nerve cells based on the presence or absence of co-localization of granular TDP-43 signals and granular stress granule marker signals in the cytoplasm.

2. The analysis method according to claim 1, wherein the stress granule marker is at least one selected from the group consisting of G3BP1, G3BP2, ATXN2, ATXN2L, UBAP2, UBAP2L, TIAL1, TIA1, TIAR, CARPIN1, USP10, TRIM25, TRIM56, DDX3X, FMR1, CSDE1, PRRC2C, NUFI2, and HDAC6.

3. The analytical method according to claim 1, wherein the stress granule marker is at least one selected from the group consisting of G3BP1, G3BP2, ATXN2, TIAL1, and TIA1.

4. The analytical method according to any one of claims 1 to 3, wherein the analytical step for analyzing the localization of TDP-43 in the nerve cells is to analyze at least one of the following indicators: (i) the percentage of cells in which granular TDP-43 signals and granular stress granule marker signals co-localize in the cytoplasm; (ii) the number of granular TDP-43 signals co-localized with stress granule markers in the cytoplasm per cell; and (iii) the area of ​​granular TDP-43 signals co-localized with stress granule marker signals in the cytoplasm per cell.

5. The analytical method according to any one of claims 1 to 3, wherein the nerve cells are nerve cells differentiated from human-derived pluripotent stem cells.

6. The analytical method according to any one of claims 1 to 3, wherein the nerve cells are nerve cells obtained by culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death, and wherein TDP-43 aggregation is induced in the cytoplasm.

7. A kit for performing the analytical method according to any one of claims 1 to 3, comprising an anti-TDP-43 antibody and an antibody that recognizes a stress granule marker.

8. A screening method for the prevention and / or treatment of neurodegenerative diseases, comprising: a test substance contact step of bringing a test substance into contact with nerve cells; an analysis step of measuring the amount of TDP-43 localized in the cytoplasm of the nerve cells after the test substance contact step by the analysis method described in any one of claims 1 to 3; and a selection step of selecting the test substance as a candidate for the prevention and / or treatment of neurodegenerative diseases based on the amount of TDP-43 localized in the analysis step.

9. The screening method according to claim 8, wherein, in the selection step, nerve cells in which the localization of TDP-43 in the cytoplasm is reduced compared to the case in which the test substance is not in contact are selected as candidates for the prevention and / or treatment of neurodegenerative diseases.

10. The screening method according to claim 8, wherein the nerve cells are nerve cells differentiated from human-derived pluripotent stem cells.

11. The screening method according to claim 8, wherein the nerve cells are nerve cells obtained by culturing nerve cells differentiated from human-derived pluripotent stem cells in a culture medium containing a protein synthesis inhibitor at a concentration that does not induce cell death, and wherein TDP-43 aggregation is induced in the cytoplasm.

12. The screening method according to claim 8, wherein the neurodegenerative disease is at least one selected from the group consisting of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, familial frontotemporal dementia parkinsonism linked to chromosome 17, Paget's disease of bone and inclusion body myopathy with frontotemporal dementia, Perry syndrome, Parkinson's disease, Parkinson's dementia complex, familial British dementia, Huntington's disease, Machado-Joseph disease, Alzheimer's disease, Lewy body dementia, corticobasal degeneration, argyrophilic granule disease / argyrophilic granule dementia, diffuse neurofibrillary tangle disease with calcification, and progressive supranuclear palsy.

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