WO2018066701A1 - Agent prophylactique ou thérapeutique pour la dlft et sa méthode de dépistage - Google Patents

Agent prophylactique ou thérapeutique pour la dlft et sa méthode de dépistage Download PDF

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WO2018066701A1
WO2018066701A1 PCT/JP2017/036520 JP2017036520W WO2018066701A1 WO 2018066701 A1 WO2018066701 A1 WO 2018066701A1 JP 2017036520 W JP2017036520 W JP 2017036520W WO 2018066701 A1 WO2018066701 A1 WO 2018066701A1
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tau
test substance
cells
ftld
cell
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治久 井上
恵子 今村
真人 樋口
成彦 佐原
哲也 須原
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国立大学法人京都大学
国立研究開発法人量子科学技術研究開発機構
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation
    • C12N2529/10—Stimulation by light

Definitions

  • the present invention relates to a novel therapeutic agent for frontotemporal lobar degeneration. More specifically, the present invention relates to a preventive or therapeutic agent for frontotemporal lobar degeneration, comprising an inhibitor of calcium ion influx into cells.
  • neurodegenerative diseases due to the gradual increase in life expectancy and a dramatic increase in the prevalence of these diseases, late-onset neurodegenerative diseases are increasing the burden on society. Based on accumulated evidence, most neurodegenerative diseases (non-patent literature 1), including neurodegenerative tauopathy (non-patent literature 1-5), which is a disease associated with pathological aggregation of tau, a microtubule-related protein in the brain. Misfolded proteins are strongly associated as causative agents of 6-10). Mutations in the tau gene (MAPT) cause a familial disease known as frontotemporal lobar degeneration tauopathy or FTLD-Tau, which presents with dementia and is prominent in the frontal and temporal lobes of the human brain Characterized by atrophy.
  • FTLD-Tau frontotemporal lobar degeneration tauopathy
  • Tau is an axon microtubule-related protein that contributes to microtubule stabilization.
  • Alternative splicing of exon 10 encoding a microtubule binding domain (MBD) generates 3 repeat tau with 3 MBDs or 4 repeat tau with 4 MBDs.
  • Mutations in exons of MAPT are known to weaken the ability of tau to bind to microtubules, promote self-assembly, and change MAPT splicing (Non-Patent Documents 11 to 13).
  • Intron mutations in MAPT have been shown to affect splicing of exon 10 and increase 4 repeat tau accumulated in the postmortem brain of patients with intron mutations (Non-Patent Documents 14 and 15).
  • FTLD induced pluripotent stem cell
  • an object of the present invention is to elucidate one end of a mechanism involved in neurodegeneration in nerve cells of FTLD-Tau patients and provide a novel preventive or therapeutic means for frontotemporal lobar degeneration based on the mechanism. That is.
  • Another object of the present invention is to provide a method for screening a preventive or therapeutic agent for frontotemporal lobar degeneration using the mechanism.
  • the present inventors have introduced a mutation in the intron or exon MAPT (Mackenzie, IR, et al. Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update. Acta Neuropathol 119, 1-4 (2010)).
  • the FTLD-Tau iPS cell model was established using neuronal differentiation induced by Neurogenin2 (Ngn2) from FTLD-Tau patients with any of the above. This direct conversion method produces a stable amount of cortical neurons.
  • FTLD-Tau FTLD-Tau iPS cells that constitutively express DREADD (designer receptors exclusivelyactivated byrugdesigner drug). It was found to contribute to neurodegeneration.
  • DREADD designer receptors exclusivelyactivated byrugdesigner drug
  • a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising an inhibitor of calcium ion influx into cells.
  • the calcium ion influx inhibitor is an antagonist or expression inhibitor for NMDA type glutamate receptor, AMPA type glutamate receptor, group I metabotropic glutamate receptor or voltage-dependent calcium channel,
  • Agent is an agonist for a group II metabotropic glutamate receptor or a group III metabotropic glutamate receptor.
  • [5] The agent according to any one of [1] to [4], which is an inhibitor of tau protein misfolding.
  • [6] The agent according to any one of [1] to [5], wherein the frontotemporal lobar degeneration is frontotemporal lobar degeneration tauopathy (FTLD-Tau).
  • FTLD-Tau frontotemporal lobar degeneration tauopathy
  • [7] The agent according to [6], wherein the frontotemporal lobar degeneration tauopathy is caused by a MAPT gene mutation.
  • a screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising the following steps.
  • a step of bringing a test substance into contact with a cell (2) a step of measuring the inflow of calcium ions into the cell, (3) a step of comparing the degree of inflow of calcium ions in (2) above with the degree of inflow of calcium ions in cells not contacted with the test substance; (4) a step of adding a test substance having a reduced level of calcium ion influx to a nerve cell associated with a pathological condition of FTLD and detecting or measuring the accumulation or release of misfolded tau protein, and (5) A step of selecting the test substance as a candidate for a prophylactic or therapeutic agent for frontotemporal lobar degeneration when accumulation of misfolded tau protein or release to the outside of the cell decreases.
  • a screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising the following steps. (1) A step of bringing a test substance into contact with a nerve cell associated with a pathological condition of FTLD, (2) a step of measuring the inflow of calcium ions into the cells and the degree of tau protein misfolding, (3) a step of comparing the degree of calcium ion influx and tau protein misfolding in (2) above with the degree of calcium ion influx and tau protein misfold in cells not contacted with the test substance; 4) A step of selecting a test substance that has reduced both the inflow of calcium ions and the degree of tau protein misfolding as a candidate for a prophylactic or therapeutic agent for frontotemporal lobar degeneration.
  • a screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising the following steps. (1) a step of contacting a test substance with a cell in which the exogenous muscarinic receptor subtype M4 is constitutively expressed; (2) a step of measuring the amount of cAMP in the cell, (3) The step of comparing the amount of cAMP in (2) above with the amount of intracellular cAMP that was not contacted with the test substance, and (4) the test substance with a reduced cAMP amount was determined for frontotemporal lobar degeneration. Step of selecting as a prophylactic or therapeutic agent candidate [11] A screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration, comprising the following steps.
  • IP3 concentration or DAG concentration in (2) above with the intracellular IP3 concentration or DAG concentration not contacted with the test substance, and (4) the test substance with reduced IP3 concentration or DAG concentration
  • a candidate for a prophylactic or therapeutic agent for frontotemporal lobar degeneration [12] A method for preventing or treating frontotemporal lobar degeneration in a mammal, comprising administering an effective amount of an inhibitor of calcium ion influx into cells to a mammal.
  • An inhibitor of calcium ion influx into cells for use in the
  • FIG. 1 shows the generation of iPS cells from FTLD-Tau patients with either intron or exon MAPT mutations, and gene editing for isogenic control.
  • the FTLD-Tau1 iPS cell line carried a MAPT intron mutation (intron 10 + 14C ⁇ T), and the FTLD-Tau2 iPS cell line carried a MAPT exon mutation (R406W). .
  • FTLD-Tau1 corrected The mutation of (c, d) FTLD-Tau1 was corrected using CRISPR / Cas9 system (FTLD-Tau1 corrected). Sanger sequence analysis revealed the correction of mutations after gene editing.
  • RT-PCR showing FTLD-Tau1 neurons with increased expression of 4 repeat tau and FTLD-Tau1 corrected neurons with normal expression ratio of 4 repeat tau and 3 repeat tau. A full-length image of the gel is shown in FIG. 8 (a).
  • Vaginal cell lysates were dephosphorylated with lambda phosphatase, separated by SDS-PAGE for comparison with recombinant tau ladder, and then detected using anti-total tau antibody (Tau12) .
  • FIG. 8 shows misfolded tau accumulation in FTLD-Tau neurons.
  • FIG. 8 shows misfolded tau accumulation in FTLD-Tau neurons.
  • FIG. 8 shows misfolded tau accumulation in FTLD-Tau neurons.
  • FIGS. 8 (c) -8 (f) Full length images of the blots are shown in FIGS. 8 (c) -8 (f).
  • FIG. 3 shows calcium dysregulation and neuronal loss in FTLD-Tau neurons.
  • (a, b) Calcium imaging after electrical stimulation. Intracellular Ca 2+ levels increased by electrical stimulation were higher in FTLD-Tau neurons than in controls (n 6; one-way ANOVA, p ⁇ 0.05; post hoc test, * p ⁇ 0.05).
  • (c, d) Immunofluorescence staining of ⁇ III tubulin and NeuN on days 8 and 21 in control and FTLD-Tau neurons. Survival of FTLD-Tau neurons from day 8 to day 21 was reduced compared to control neurons (n 6; one-way ANOVA, p ⁇ 0.05; post hoc test, * p ⁇ 0.05 ).
  • FIG. 3 shows calcium dysregulation and neuronal loss in FTLD-Tau neurons.
  • FIG. 1 shows the characterization of iPS cells and the generated neurons.
  • iPS cells show normal karyotype.
  • FIG. 6 relates to FIG. 1 and shows an electrophysiological analysis of iPS cell-derived neurons. To confirm whether iPS cell-derived neurons are functional, the electrophysiological properties of 3 weeks differentiated neurons derived from control iPS cells were evaluated by patch clamp analysis.
  • the membrane potential was recorded from a resting membrane potential of -60 mV, following a 75 pA depolarization current injection. The continuity of action potential was recorded by current injection.
  • Neurons derived from iPS cells showed paired-pulse depression in response to paired pulse stimulation.
  • FIG. 8 shows a full-length image of the gel and blot shown in the main figure in relation to FIGS. 1, 2 and 4.
  • A Full length image of the gel shown in FIG. 1 (e).
  • Agent of the present invention provides a preventive or therapeutic agent for frontotemporal lobar degeneration, comprising an inhibitor of calcium ion influx into cells (hereinafter abbreviated as “agent of the present invention”). May be).
  • the “calcium ion influx inhibitor” of the present invention means a drug in which the inflow of calcium ions into cells is reduced before the drug is introduced or compared with a control group to which no drug is administered.
  • FTLD Frontotemporal lobar degeneration
  • FTLD pathological classification includes frontotemporal lobar degeneration tauopathy (FTLD-tau), FTLD-TDP, FTLD-FUS, FTLD-UPS, and FTLD-without inclusion.
  • the FTLD patient to whom the calcium ion influx inhibitor is administered is not particularly limited, but is desirably an FTLD-tau patient, and particularly preferably an FTLD patient with a MAPT gene mutation.
  • mutations in introns of MAPT for example, mutations in intron 10 + 14C ⁇ T, in which cytosine, the 14th base of intron 10 of the MAPT gene, is replaced with thymine
  • FTLD-Tau patients with abnormally folded (misfolded) tau protein accumulation due to (mutation) etc. are particularly desirable.
  • compositions When using calcium ion inflow inhibitors as pharmaceuticals, they are used as is or known pharmaceutically acceptable carriers (excipients, diluents, extenders, binders, lubricants, flow aids, disintegrants, surfactants). Or a conventional additive and the like, and can be prepared as a pharmaceutical composition.
  • the pharmaceutical composition is prepared in an orally administered form such as tablets, pills, capsules, powders, granules, syrups, emulsions and suspensions; injections, drops, external preparations, suppositories, etc. Depending on the parenteral administration agent, etc., it can be systemically or locally administered orally or parenterally.
  • intravenous administration In the case of parenteral administration, intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, and the like are possible.
  • the above-mentioned appropriate dosage form can be produced by blending the active ingredient with an acceptable ordinary carrier, excipient, binder, stabilizer, diluent and the like.
  • acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.
  • the dose varies depending on the type of active ingredient, the route of administration, age of the subject or patient, body weight, symptoms, etc., and cannot be specified unconditionally.
  • About mg to 2 g, preferably about 5 mg to several tens mg can be administered once to several times a day. In the case of injection, it is sufficient to administer about 0.1 mg to about 500 mg as an active ingredient in an adult, and the daily dose can be administered once or divided into several times.
  • ⁇ G i / o signaling In one embodiment of the invention, inhibition of calcium ion influx is effected via Gi signaling.
  • ⁇ G i / o signaling '' means that G protein G i / o is shared to inhibit adenylyl cyclase, reduce cAMP production, and downstream protein kinase A (PKA)
  • PKA protein kinase A
  • the term “performed via Gi signaling” means that activation of Gi / o or inhibition of influx by inhibiting adenylate cyclase or PKA. means.
  • inhibitors of adenylate cyclase include Adenosine 3 ', 5'-cyclic Monophosphate, 8- (4-Chlorophenylthio)-, Sodium Salt 116812 (8-CPT-cAMP, Na), Adenosine 3', 5'- cyclic Monophosphate, 8-Bromo-, Sodium Salt 203800 (8-Bromo-cAMP, Na), Adenylyl Cyclase Toxins Inhibitor 116845 (Ethyl-5-aminopyrazolo [1,5-a] quinazoline-3-carboxylate), Adenylyl Cyclase Type V Inhibitor, NKY80 116850 [2-Amino-7- (furanyl) -7,8-dihydro-5 (6H) -quinazolinone], 2 ', 5'-Dideoxyadenosine 288104 (2', 5'-dd-Ado), MDL -12,330A, Hydro
  • PKA inhibitors include, but are not limited to, H89, KT5720, ML-9, K-252a, PKI (6-22) amide, and the like. Further, an antibody or aptamer against adenylate cyclase or PKA may be used.
  • NMDA type glutamate receptor NMDA receptor
  • AMPA type glutamate receptor AMPA type glutamate receptor
  • mGluR metabotropic glutamate receptor
  • voltage-dependent calcium Examples include channels (VGCC) and calcium release channels existing on the surface of the endoplasmic reticulum (ER).
  • NMDA-type glutamate receptors mediate calcium influx in postsynaptic cells on dendritic spines that are present in various neurons and cortex.
  • AMPA-type glutamate receptors are ion-coupled receptors, and are known to localize mainly in GABAergic neurons that are almost free of spines and mediate calcium influx.
  • Metabotropic glutamate receptor is a type of G protein-coupled receptor (GPCR) that is known to be widely distributed in the central and peripheral nervous systems. It is classified into three types, II and Group III. Group I mGluR is mainly localized in the postsynaptic membrane, and when it receives a signal, it couples with G aq to activate PLC, and the intracellular diacylglycerol (Diacylglycerol) and inositol triphosphate (Inositol triphosphate) Increase concentration. As a result, PKC is activated, and Ca 2+ stored in the endoplasmic reticulum is released into the cell to increase the intracellular calcium concentration.
  • GPCR G protein-coupled receptor
  • Group II mGluR and Group III mGluR are mainly localized in the presynaptic membrane and inhibit adenylate cyclase activity, thereby inhibiting calcium ion influx into cells.
  • VGCC is a channel that opens in a voltage-dependent manner by depolarization of membrane potential, and calcium ions flow into nerve cells by opening.
  • VGCC forms a complex composed of multiple subunits, and its structure is rich in diversity.
  • On the surface of the endoplasmic reticulum there are calcium release channels such as inositol triphosphate receptor (IP3R) and ryanodine receptor (RyR).
  • the term “agonist” means a substance that binds to a specific receptor and induces a response in a cell
  • positive allosteric modulators (PAM) Positive Allosteric Modulator
  • An allosteric modulator means a molecule that binds to an allosteric site and enhances or inhibits the binding or signal transduction of an orthosteric ligand to exert its action.
  • An allosteric modulator is an orthosteric ligand.
  • PAM Physical Allosteric Modulator
  • NAM Negative Allosteric Modulator
  • SAM Neutral Allosteric Modulator
  • Antagonist means a substance that decreases, inhibits or prevents the action of another molecule or the activity of a receptor site.
  • Antagonists also include, but are not limited to, competitive antagonists, non-competitive antagonists, uncompetitive antagonists, inhibitors of ion channels, NAMs.
  • a competitive antagonist binds reversibly to the receptor at the same binding site (active site) as the endogenous ligand or agonist, but does not activate the receptor.
  • Non-competitive antagonists also known as allosteric antagonists
  • Non-competitive antagonists do not compete with agonists for binding.
  • the bound antagonist may reduce the affinity of the receptor agonist or prevent changes in the receptor conformation that are required for receptor activation after agonist binding.
  • Uncompetitive antagonists differ from non-competitive antagonists in that they require receptor activation by an agonist before binding to an allosteric binding site.
  • antibodies, aptamers, dominant negative mutants and the like that inhibit the function of receptors and calcium channels are also included as antagonists.
  • NMDA receptor antagonists include AP5 (APV, R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoic acid), CPPene (3-[(R) -2-carboxypiperazin-4-yl ] -prop-2-enyl-1-phosphonic acid), Selfotel, Amantadine, Atomoxetine, AZD6765, Agmatine, Chloroform, Dextrallorphan, Dextromethorphan, Dextrorphan, Diphenidine, Dizocilpine (MK-801), Ethanol, Eticyclidine, Gacyclidine, Ibogaine, Magnesium , Memantine, Methoxetamine, Nitromemantine, Nitrous oxide, Phencyclidine, Rolicyclidine, Tenocyclidine, Methoxydine, Tiletamine, Neramexane, Eliprodil, Etoxadrol, Dexox
  • AMPA receptor antagonists include CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), DNQX (6,7-Dinitroquinoxaline-2,3-dione), NBQX (2,3-Dioxo-6-nitro -1,2,3,4-tetrahydrobenzo [f] quinoxaline-7-sulfonamide), Kynurenic acid, Ethanol, GYKI-52466 (4- (8-Methyl-9H-1,3-dioxolo [4,5-h] [2,3] benzodiazepin-5-yl) -benzenamine hydrochloride), GYKI-53655 (1- (4-Aminophenyl) -3-methylcarbamyl-4-methyl-3,4-dihydro-7,8-methylenedioxy-5H- 2,3-benzodiazepine hydrochloride), Perampanel, Talampanel, etc., but are not limited thereto.
  • Voltage-gated calcium channel antagonists include Amlodipine (Norvasc), dihydropyridine (DHP), phenylalkylamine (PAA), benzothiazepine (BTZ), Aranidipine (Sapresta), Azelnidipine (Calblock), Barnidipine (HypoCa), Benidipine (Coniel), Cilnidipine (Atelec, Cinalong, Siscard), Clevidipine (Cleviprex), Isradipine (DynaCirc, Prescal), Efonidipine (Landel), Felodipine (Plendil), Lacidipine (Motens, Lacipil), Lercanidipine (Zanidip), Manidipine (Calslot, cardipine) (Cardene, Carden SR), Nifedipine (Procardia, Adalat), Nilvadipine (Nivadil), Nimodipine
  • Group I mGluR antagonists include JNJ-16259685, R-214127, YM-202074, YM-230888, YM-298198, FTIDC, A-841720, Cyclothiazide, Lithium, LY-344545, Mavoglurant, Remeglurant, SIB-1893, Basimglurant , Dipraglurant, Fenobam, GRN-529, MPEP, MTEP and Raseglurant, but are not limited thereto.
  • Group II mGluR agonists include MGS-0028, LY404040, LY379268, LY354740, (R) -2-amino-4- (4-hydroxy [1,2,5] thiadiazol-3-yl) butyric acid, (S) -aminoadipic acid, JNJ-46356479, JNJ-40411813, GSK-1331258, Imidazo [1,2-a] pyridines, 3-Aryl-5-phenoxymethyl-1,3-oxazolidin-2-ones, 3- (Imidazolyl methyl) -3-aza-bicyclo [3.1.0] hexan-6-yl) methyl ethers, BINA and LY-487379, but are not limited thereto.
  • Group III mGluR agonists include Cinnabarinic acid, LSP1-2111, LSP4-2022, L-AP4 (L-2-amino-4-phosphonobutyrate), ML-128, VU-0418506, VU-001171, VU-0155041, PHCCC , ADX88178, (S) -aminoadipic acid, AMN082 (N, N'-dibenzhydrylethane-1,2-diamine dihydrochloride), (S) -3,4-DCPG ((S) -3,4-Dicarboxyphenylglycine) and AZ 12216052 (2-[[(4-Bromophenyl) methyl] thio] -N- [4- (1-methylpropyl) phenyl] acetamide) and the like, but is not limited thereto.
  • Antisense nucleic acids, siRNA, shRNA, miRNA, ribozyme, antigene, etc. for receptors etc. are designed appropriately using known design software based on the base sequence of genes such as known receptors and mRNA, etc. It can be easily synthesized using an automatic DNA / RNA synthesizer.
  • the present invention also provides a screening method for a preventive or therapeutic agent for frontotemporal lobar degeneration (hereinafter sometimes abbreviated as “method of the present invention”), comprising the following steps. (1) A step of bringing a test substance into contact with a cell, (2) a step of measuring the inflow of calcium ions into the cell, (3) a step of comparing the degree of inflow of calcium ions in (2) above with the degree of inflow of calcium ions in cells not contacted with the test substance; (4) a step of adding a test substance having a reduced level of calcium ion influx to a nerve cell associated with a pathological condition of FTLD and detecting or measuring the accumulation or release of misfolded tau protein, and (5) When the accumulation or release of misfolded tau protein is reduced before or after the addition of the test substance, the test substance is removed from frontotemporal lobar degeneration. Selecting as a candidate for a prophylactic or therapeutic agent.
  • a screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising the following steps. (1 ′) a step of bringing a test substance into contact with a neuron with a pathological condition of FTLD, (2 ′) a step of measuring the inflow of calcium ions into the cells and the degree of tau protein misfolding, (3 ′) comparing the degree of calcium ion influx and tau protein misfolding in (2 ′) with the degree of calcium ion influx and tau protein misfold in cells not contacted with the test substance, And (4 ′) a step of selecting a test substance that has reduced both the influx of calcium ions and the degree of tau protein misfolding as a candidate for a prophylactic or therapeutic agent for frontotemporal lobar degeneration.
  • the nerve cell with the pathological condition of FTLD may be a nerve cell collected from an FTLD patient, or a pluripotent stem cell (iPS cell) is prepared using a somatic cell collected from an FTLD patient. It may be a nerve cell obtained by differentiating a cell.
  • iPS cells can be appropriately produced by methods known per se using somatic cells collected from FTLD patients. As a method for inducing differentiation of iPS cells into neurons, various known differentiation induction methods can be appropriately selected and used.
  • a method of forcibly expressing Ngn2 in cells and differentiating into cortical neurons can be mentioned. It is done.
  • the FTLD patient is not particularly limited, but an FTLD-Tau patient having a specific mutation in the MAPT gene as described above is particularly desirable.
  • test substance used in the screening method of the present invention is not particularly limited, and examples thereof include proteins, peptides, nucleic acids, inorganic compounds, natural or synthetically prepared organic compounds, and the like.
  • the test substance is a peptide library of 3 to 50 amino acids, preferably 5 to 20 amino acids, or a molecular weight of 100 to 2000, preferably prepared using combinatorial chemistry techniques known to those skilled in the art.
  • Can include 200 to 800 low molecular weight organic compound libraries.
  • concentration of the test substance to be brought into contact with the cell is not particularly limited, and is usually about 0.1 ⁇ M to about 100 ⁇ M, preferably 1 ⁇ M to 50 ⁇ M.
  • the time for contacting the cell and the test substance is not particularly limited and is set as appropriate. For example, it is about 5 to 30 minutes, preferably about 10 to 20 minutes.
  • the test substance can be used by appropriately dissolving or suspending in a solvent such as water, a buffer such as phosphate buffer or Tris buffer, ethanol, acetone, dimethyl sulfoxide, or a mixture thereof.
  • the inflow of calcium ions into the cell and the accompanying membrane potential depolarization can be measured by well-known methods such as the calcium imaging method and the patch clamp method, respectively.
  • the inflow of calcium ions into the cell can be detected using, for example, a calcium fluorescent probe.
  • the calcium fluorescent probe that can be used in the present invention includes 1- [6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methyl).
  • Examples include, but are not limited to, phenoxy) ethane-N, N, N ′, N′-tetraacetic acid, pentaacetoxymethyl ester (Fura 2-AM), and the like.
  • the membrane potential can be measured, for example, by a nerve recording method such as a microelectrode method or a patch clamp method, or may be measured using a membrane potential measurement fluorescent probe.
  • Fluorescent probes for membrane potential measurement include 4- (4- (didecylamino) styryl) -N-methylpyridinium iodide (4-Di-10-ASP), bis- (1,3-dibutylbarbituric acid trimethine oxo Nord (DiSBAC2 (3)), 3,3′-dipropylthiadicarbocyanine iodide (DiSC3 (5)), 5,5 ′, 6,6′-tetrachloro-1,1 ′, 3,3 ′ -Tetraethylbenzimidazolyl carbocyanine iodide (JC-1) and rhodamine 123, but are not limited to these.
  • misfolded tau protein is immunostained using, for example, an antibody that specifically recognizes the protein (for example, TOC1 antibody), or dot blot analysis, as shown in the examples below. However, it is not limited to these methods as long as misfolded proteins can be detected or measured.
  • a screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising the following steps.
  • D the test substance with a decreased amount of cAMP Selecting as a prophylactic or therapeutic agent candidate.
  • a screening method for a prophylactic or therapeutic agent for frontotemporal lobar degeneration comprising the following steps is also provided.
  • A contacting the test substance with a cell in which the exogenous muscarinic receptor subtype M3 is constitutively expressed;
  • B measuring the amount of inositol trisphosphate (IP3) or diacylglycerol (DAG) in the cell;
  • C The step of comparing the amount of IP3 or DAG in (b) above with the amount of intracellular IP3 or DAG that was not contacted with the test substance, and (d) the test substance in which the amount of IP3 or DAG was reduced Is selected as a candidate for a prophylactic or therapeutic agent for frontotemporal lobar degeneration.
  • the method described above may further include a step corresponding to step (4) of the method of the present invention in order to confirm the therapeutic effect of the candidate substance more reliably.
  • Constant expression of exogenous muscarinic receptor subtype M3 or M4 should be carried out by introducing into the cell an expression vector containing nucleic acids whose sequences encoding these receptor subtypes are known per se. Can do.
  • expression vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV). , PcDNAI / Neo) and the like.
  • Intracellular cAMP is measured, for example, by incubating the cells in the presence and absence of the test substance for an appropriate period of time and then performing a competitive immunoassay on the extract obtained by disrupting the cells. Can do. It can also be evaluated by labeling cells with [ 3 H] adenine and measuring the radioactivity of the produced [ 3 H] cAMP, but any other known method can be used. In another embodiment, there is a method for evaluating the amount of cAMP by measuring the expression level of a reporter gene under the control of a cAMP response element (CRE).
  • CRE cAMP response element
  • cells introduced with a vector containing an expression cassette linked to a DNA encoding a reporter protein downstream of a promoter containing CRE are cultured for an appropriate time in the presence and absence of a test substance.
  • the amount of intracellular cAMP is evaluated by measuring and comparing the expression of the reporter gene in the extract obtained by crushing using a known technique.
  • IP3 content Measurements of inositol triphosphate amount of intracellular added [3 H] inositol in the cell, or measuring the generated [3 H] inositol 3 radioactive phosphate, measure Ca 2+ of intracellular You can also evaluate it.
  • the measurement can be performed using a fluorescent probe that specifically binds to IP3, but any other known method can be used.
  • the DAG amount can be measured, for example, by the following method. Using the cell extract sample, phosphatidine produced by phosphorylating DAG of the sample with a kinase is hydrolyzed by lipase into glycerol-3-phosphate. Subsequently, glycerol-3-phosphate oxidase is oxidized with glycerol-3-phosphate oxidase, and the resulting hydrogen peroxide is reacted with a fluorescent probe that reacts specifically with hydrogen peroxide, and excited at 530-560 nm / fluorescence By detecting at 585-595 nm, the amount of DAG in the sample can be measured. Alternatively, measurement may be performed using a DAG fluorescent probe, but any other known method may be used.
  • iPS cells As described before the preparation and cell culture of iPS cells (Okita, K., et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.Stem Cells 31, 458-466 (2013).), Epithelial vectors for OCT3 / 4, Sox2, Klf4, L-Myc, Lin28, p53-shRNA and EBNA1, and human iPS cells with MAPT mutations from fibroblasts or monocytes And cultured on a SNL feeder layer using a human iPS cell medium (ReproCELL) supplemented with 4 ng / ml basic FGF (Wako Chemicals) and penicillin / streptomycin. As a control, 201B7 iPS cell line (Takahashi, K., et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007).) was used.
  • a guide RNA was designed to target the 5′-GCAACGTCCAGTCCAAGTGTGG-3 ′ (SEQ ID NO: 1) site. Oligonucleotides of guide RNA were ligated to human H1 polymerase III promoter (Li, HL, et al. Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.
  • NEPA21 electroporator (Nepagene) was applied to 1 million iPS cells, 10 ⁇ g pHL-Hi guide RNA expression plasmid, 10 ⁇ g pHL-EF1a-hcSpCas9 plasmid, and 10 ⁇ g donor The gene was introduced by electroporation with the plasmid. Four days after gene transfer, puromycin selection was performed for 10 days.
  • Puromycin resistant colonies were selected and expanded for genomic DNA extraction and genotyping by PCR using the following primers: forward primer, 5'-AAATGCAGTCGTGGGAGACC-3 '(SEQ ID NO: 2); reverse primer, 5' -GAGTCCCGAATCTCACGGAGACA-3 '(SEQ ID NO: 3). All amplified PCR bands were further analyzed by Sanger sequencing to confirm that there were no sequence changes in the homology arms.
  • the transposase vector pHL-EF1 ⁇ -hcPBase (Matsui, H., et al. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.
  • PCR of the puromycin cassette was performed and non-integration was confirmed using the following primers: forward primer, 5'-CTGCTGCAACTTACCTCCGGGATG-3 '(SEQ ID NO: 4 ); Reverse primer, 5′-CCAATCCTCCCCCTTGCTGTCCTG-3 ′ (SEQ ID NO: 5).
  • iPS cell lines are shown in Table 1.
  • piggyBac vector for expression of Ngn2 and introduction into iPS cells
  • Ngn2 transcription factor was introduced into mouse or human iPS cells using the piggyBac vector.
  • This vector containing the Ngn2 and neomycin resistance genes under the control of the tetracycline operator rtTA is the KW110_PB_TA_ERN (Ef1a_rtTA_neo) vector backbone (Kim, SI, et al. Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons. Methods Mol Biol 1357, 111-131 (2015).).
  • the prepared vector was co-introduced into iPS cells using Lipofectamine LTX (Invitrogen) together with the pCyL43 vector encoding transposase. After clone selection with neomycin, iPS cells harboring a tetracycline-inducible Ngn2 construct were established.
  • Preparation of iPS cell-derived cortical neurons by induction of Ngn2 iPS cells were dissociated into single cells using actase, and a 1: 1 ratio of DMEM / F12 (Life Technologies) and Neurobasal (Life Technologies), 1% N2 supplement, 2% B27 supplement, 10 ng / ml brain-derived neurotrophic factor (BDNF, R & D Systems), 10 ng / ml glial cell-derived neurotrophic factor (GDNF, R & D Systems) and 10 ng / ml neurotrophin-3 (NT-3; R & D Systems) was used to seed on plastic plates or coverslips coated with matrigel with 1 ⁇ g / ml doxycycline (Clontech).
  • BDNF brain-derived neurotrophic factor
  • GDNF ng / ml glial cell-derived neurotrophic factor
  • NT-3 10 ng / ml neurotrophin-3
  • the tol2 vector containing M4D and hygromycin resistance gene under CAG promoter control was transformed into pT2AL200R175-CAGGS-EGFP vector backbone (Kawakami, K. & Noda, T. Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics 166, 895-899 (2004).).
  • the prepared vector was simultaneously introduced into iPS cells into which Ngn2 had been introduced together with a vector encoding transposase using Lipofectamine LTX. After clone selection using hygromycin, an iPS cell line carrying M4D was established.
  • the prepared iPS cells were differentiated into neurons with doxycycline and nerve medium for 8 days, and then 100 nM pharmacologically inactive designer drug clozapine-N-oxide (CNO) was added to the culture medium to add DREADD. I was stimulated. On the 14th day, Western blot analysis and dot blot analysis were performed.
  • CNO pharmacologically inactive designer drug clozapine-N-oxide
  • RNA of cultured cells was extracted using RNeasy Plus Mini kit (QIAGEN). One microgram of RNA was reverse transcribed using ReverTra Ace (TOYOBO). PCR analysis was performed using TAKARA Ex Taq (TAKARA). As previously reported, primer sets for 3 repeat tau and 4 repeat tau were used: forward 5'-AAGTCGCCGTCTTCCGCCAAG-3 '(SEQ ID NO: 6); reverse 5'-GTCCAGGGACCCAATCTTCGA-3' (SEQ ID NO: 7) . RT-PCR products were evaluated on 3% agarose gels: 3 repeat tau and 4 repeat tau RT-PCR products were 288 and 381 bp, respectively (Iovino, M., et al. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations. Brain 138, 3345-3359 (2015).).
  • RNA of the cultured cells on day 7 was extracted using the RNeasy Plus Mini kit (QIAGEN). One microgram of RNA was reverse transcribed using ReverTra Ace (TOYOBO). Quantitative PCR analysis was performed by reverse transcription reaction with SYBR Premix Ex Taq II (TAKARA) using Step One Plus (Applied Biosystems). The primer set is shown in Table 2.
  • Immunocytochemical cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, washed with PBS, and permeated into PBS containing 0.2% Triton X-100 for 10 minutes at room temperature. Subsequently, blocking was performed with Block Ace (Yukijirushi) for 30 minutes. After overnight incubation with the primary antibody at 4 ° C., the cells were washed 3 times with PBS and incubated with the appropriate secondary antibody for 1 hour at room temperature.
  • Cell images were acquired from Delta Vision (Applied Precision) or IN Cell Analyzer 6000 (GE Healthcare). Cell numbers were quantified with IN Cell Analyzer 6000 and IN CELL Developer toolbox software 1.9 (GE Healthcare).
  • ⁇ III tubulin (1: 2,000, Covance), NeuN (1: 500, Millipore), TOC1 (1: 1,000), Nanog (1: 200, Abcam), SSEA4 ( 1: 500, Millipore).
  • the total protein extract (20 ⁇ g per lane) is separated by size on a 10% polyacrylamide gel and transferred to an Immobilon-P membrane (Millipore). did. Membranes were blocked with 5% skim milk, hybridized with the appropriate antibody and visualized using the ECL Prime detection kit (GE Healthcare). Images were acquired from ImageQuant LAS 4000 (GE Healthcare). The following primary antibodies were used: MAP2 (1: 1,000, CST), ⁇ III tubulin (1: 5,000, Covance), Tau12 (1: 10,000, Millipore), TOC1 (1: 5,000) and ⁇ -actin (1: 5,000), Sigma).
  • the TOC1 antibody has a linear epitope displayed in the oligomer compared to the monomer or filament in the oligomer (Ward, SM, et al. TOC1: characterization of a selective oligomeric tau antibody. J Alzheimers Dis 37, 593-602 (2013).) Therefore, it is noteworthy that non-denaturing conditions are required to evaluate oligomeric tau using TOC1 antibody. The use of denaturing conditions makes no distinction between these different tau species, oligomers, monomers and / or filaments using TOC1. Table 3 shows the tau antibodies used in this paper.
  • the membrane was blocked with 5% skim milk, hybridized with the appropriate antibody, and visualized using Western Lightning Plus-ECL (PerkinElmer). Images were acquired from ImageQuant LAS 4000 (GE Healthcare). The following primary antibodies were used: Tau12 (1: 10,000, Millipore), Tau46 (1: 1,000), TOC1 (1: 5,000).
  • Cell viability assay Dissociated iPS cells transfected with Ngn2 into single cells, 5 ⁇ 10 4 cells / well on a 96-well plate (BD Bioscience) coated with Matrigel using nerve medium containing 1 ⁇ g / ml doxycycline Sowing. Cells were fixed and stained on days 8 and 21. The number of viable neurons stained with NeuN was quantified with IN Cell Analyzer 6000 (GE Healthcare) and expressed as the number of neurons on day 21/8. D-2-amino-5-phosphonopentanoate (AP-5) (SIGMA) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (SIGMA) from day 8 to day 21 The number of surviving neurons was evaluated on day 21.
  • AP-5 SIGMA
  • CNQX 6-cyano-7-nitroquinoxaline-2,3-dione
  • PI stained cells were incubated with 1.5 ⁇ M PI (Nacalai Tesque) for 30 minutes at 37 ° C. and fixed with 4% paraformaldehyde. The number of PI stained cells was quantified by IN Cell Analyzer 6000.
  • Enzyme-linked immunosorbent assay (ELISA) of oligomeric tau A 96-well plate (Greiner) was coated overnight at 4 ° C. with 3 ⁇ g / ml TOC1 antibody in 0.05 M sodium carbonate buffer. After washing and blocking with TBS-T containing 1% BSA, 100 ⁇ l of culture medium was added and incubated at room temperature for 2 hours. A standard curve was obtained using recombinant oligomeric tau protein. For detection, plates were incubated with 2 ⁇ g / ml affinity purified rabbit polyclonal anti-human tau antibody followed by sheep anti-rabbit IgG F (ab) ′ 2 fragment conjugated to horseradish peroxidase (1: 3,000; GE Healthcare) did.
  • iPS cells were dissociated into single cells and seeded at 5 ⁇ 10 4 cells / well on a 96-well plate (BD Bioscience) coated with Matrigel using nerve medium containing 1 ⁇ g / ml doxycycline.
  • e-FDSS / ⁇ CELL Haamamatsu Photonics
  • Electrophysiological recordings Whole cell patch clamp recordings were performed from iPS cell-derived neurons.
  • the recording micropipette was filled with an intracellular solution composed of 140 mM KCl, 2 mM MgCl 2 , 10 mM HEPES and 1 mM EGTA, adjusted to pH 7.4 with NaOH. Cells are maintained at 30 ° C throughout the experiment with an oxidized Krebs Ringer solution consisting of 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 26 mM NaHCO 3 , 1 mM MgCl 2 , 2 mM CaCl 2 and 20 mM glucose. Continuously perfused. Voltage clamp and current clamp recordings were performed using an EPC9 amplifier (HEKA) and the data were analyzed using Patchmaster software (HEKA).
  • HEKA EPC9 amplifier
  • the intron 10 + 14C ⁇ T mutation was corrected using CRISPR-Cas9 (FIGS. 1 (c) and 1 (d)).
  • Ngn2 was introduced into these iPS cells by a piggyBac vector having a tet-on expression system, followed by drug selection for stable cell line establishment.
  • the established iPS cells were converted to cortical neurons after 7 days of culture in nerve medium containing doxycycline. There was no difference in the differentiation tendency of the obtained strains.
  • the prepared neurons express mRNA of neurotransmitter receptor (Fig. 5 (c)), and their functional properties were shown by electrophysiological analysis (Fig. 6 (a) -6 (h) )). As previously reported (Hutton, M., et al.
  • FTLD-Tau neurons show typical misfolded tau spots and dots, and control neurons, including genetically modified strains, have misfolded tau spots or dots. Most were negative.
  • Dot blot analysis showed the accumulation of misfolded forms of tau in cells in a non-denatured state using the TOC1 antibody (FIGS. 2 (d), (e)). Also, as previously shown (Ward, SM, et al. TOC1: characterization of a selective oligomeric tau antibody. J Alzheimers Dis 37, 593-602 (2013)), to detect tau species under denaturing conditions. Misfolded tau was analyzed by Western blot analysis using TOC1 antibody.
  • DREADD muscarinic acetylcholine M4 receptor
  • D-2-amino-5-phosphonopentanoate (AP-5) and ⁇ -amino-3-hydroxy-5-methyl which are inhibitors of N-methyl-D-aspartate (NMDA) receptors 4-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), an inhibitor of the 4-isoxazolepropionic acid (AMPA) receptor, increased the survival of FTLD-Tau neurons (Fig. 4 (e) and 4 (f)).
  • the inflow inhibitor of calcium ions into cells of the present invention is useful for the prevention and / or treatment of frontotemporal lobar degeneration, and the screening method using the inflow of calcium ions into cells as an index is It is useful for screening for preventive and / or therapeutic agents for temporal lobe degeneration.

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Abstract

L'invention concerne un agent prophylactique ou thérapeutique pour la dégénérescence lobaire fronto-temporale (DLFT), ledit agent comprenant un inhibiteur de l'influx d'ions calcium dans des cellules. L'invention concerne également une méthode de dépistage d'un agent prophylactique ou thérapeutique pour la DLFT, ladite méthode comprenant : (1) une étape de mise en contact d'une substance à tester avec des cellules ; (2) une étape de mesure de l'influx d'ions calcium dans les cellules ; (3) une étape de comparaison du taux d'influx d'ions calcium mesuré en (2) avec un taux d'influx d'ions calcium dans des cellules non mises en contact avec la substance à tester ; (4) une étape d'ajout d'une substance à tester, qui abaisse le taux d'influx d'ions calcium, à des neurones possédant l'état pathologique de la DLFT et de détection ou de mesure de l'accumulation ou de la décharge extracellulaire de protéines tau mal repliées ; et (5) une étape, lorsque l'accumulation ou la décharge extracellulaire de protéines tau mal repliées est réduite, de sélection de la substance à tester en tant que candidat pour un agent prophylactique ou thérapeutique pour la DLFT.
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JPWO2020090966A1 (ja) * 2018-11-01 2021-09-30 国立研究開発法人国立長寿医療研究センター タウオパチーの治療薬または予防薬のスクリーニング方法およびタウオパチーの診断方法
CN114599397A (zh) * 2019-10-25 2022-06-07 国立大学法人京都大学 Tau蛋白病的预防或治疗剂

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112469827A (zh) * 2018-07-04 2021-03-09 国立大学法人东海国立大学机构 控制Tau的剪接的反义寡核苷酸及其用途
CN112469827B (zh) * 2018-07-04 2024-05-24 学校法人爱知医科大学 控制Tau的剪接的反义寡核苷酸及其用途
JPWO2020090966A1 (ja) * 2018-11-01 2021-09-30 国立研究開発法人国立長寿医療研究センター タウオパチーの治療薬または予防薬のスクリーニング方法およびタウオパチーの診断方法
JP7296136B2 (ja) 2018-11-01 2023-06-22 国立研究開発法人国立長寿医療研究センター タウオパチーの治療薬または予防薬のスクリーニング方法およびタウオパチーの診断方法
CN114599397A (zh) * 2019-10-25 2022-06-07 国立大学法人京都大学 Tau蛋白病的预防或治疗剂
CN114599397B (zh) * 2019-10-25 2024-05-28 国立大学法人京都大学 Tau蛋白病的预防或治疗剂

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