WO2017170986A1 - Agent for inducing accumulation of insolubilized tdp-43 protein, method for creating neurodegenerative disease model cells, and method for screening for therapeutic or prophylactic drug for neurodegenerative disease - Google Patents

Abstract

The purpose of the present invention is to provide the following: a method for creating neurodegenerative disease model cells, the method not depending on genetic mutation introduction and with which a morbid state can be uniformly reproduced in the nerve cells of a patient; and a screening method that uses the model cells. The present invention provides a method for creating neurodegenerative disease model cells using an agent for inducing accumulation of insolubilized TDP-43 protein, and a method for screening for a therapeutic or prophylactic drug for neurodegenerative disease using the model cells.

Description

Accumulation inducer of insolubilized TDP-43 protein, method for producing neurodegenerative disease model cell, and screening method for therapeutic or prophylactic agent for neurodegenerative disease

The present invention relates to an accumulation inducer of insolubilized TDP-43 protein, a method for producing a neurodegenerative disease model cell, and a screening method for a therapeutic or prophylactic agent for neurodegenerative disease.

In many neurodegenerative diseases, accumulation of specific proteins in nerve cells and abnormal neurites are known as pathological features, and these phenomena are thought to cause nerve cell death.

For example, in frontotemporal lobar degeneration (hereinafter referred to as FTLD) and amyotrophic lateral sclerosis (hereinafter referred to as ALS), as a pathological feature thereof, an insolubilized trans-activation response DNA binding protein-43 (trans-activation response) It has been reported that element DNA-binding protein of 43 kDa; hereinafter referred to as TDP-43 protein) accumulates in the cytoplasm (Non-patent Document 1), abnormal neurites and cell death (Non-patent Document 2). . In addition, in some patients with familial FTLD and familial ALS, it has been reported that a mutation exists in a gene encoding TDP-43 protein (hereinafter referred to as TDP-43 gene) (Non-patent Document 3).

Providing model cells that reproduce the pathological characteristics of neurodegenerative diseases is indispensable for understanding the pathological conditions of the diseases and developing therapeutic methods, and thus various model cells have been provided so far.

For example, a model that reproduces the pathological characteristics of familial FTLD and familial ALS by introducing a TDP-43 gene having a mutation into cultured cells, and a method for screening therapeutic and / or prophylactic drugs using the model are reported. (Patent Documents 1 and 2).

In recent years, attention has been focused on nerve cells obtained by differentiating human induced pluripotent stem cells (hereinafter, human iPS cells) as models of human neurodegenerative diseases. Nerve cells obtained by differentiating human iPS cells prepared from patients with neurodegenerative diseases, particularly those having mutations in the pathogenic genes of the above diseases, are expected as models that well reproduce the pathological features of the above diseases. (Patent Documents 3 and 4).

Furthermore, model cells using cancer cell-derived cell lines have also been reported (Non-patent Document 4).

International Publication No. 2009/125646 JP 2014-171425 A International Publication No. 2014/148646 Special table 2015-506905 gazette

However, more than 90% of patients with FTLD and ALS are sporadic patients who have no mutation in the TDP-43 gene or are not familial. Therefore, it is not always a sufficient model of FTLD and ALS only with model cells prepared by introducing a TDP-43 gene having a mutation found in some patients with familial diseases.

In addition, since nerve cells obtained by differentiating human iPS cells prepared from patients having mutations in the pathogenic genes of neurodegenerative diseases are also produced based on mutations in the pathogenic genes, sporadic diseases It is not a model for neurodegenerative diseases including Furthermore, when differentiating from a human iPS cell to produce a target nerve cell, it may be difficult to control the synchronization of differentiation depending on the origin of the human iPS cell. Therefore, it may be difficult to obtain a cell population exhibiting uniform pathological characteristics, which is indispensable for use in screening for therapeutic and preventive drugs for neurodegenerative diseases.

In addition, neurodegenerative diseases such as FTLD, ALS, and Alzheimer's disease, which have pathological features characterized by the accumulation of specific proteins, are considered to be human-specific diseases because there are no reports of corresponding diseases in animals. Therefore, in the past, model cells using cells derived from mammals other than humans have been difficult to sufficiently reproduce the pathological characteristics of human-specific diseases. It was considered unsuitable for screening. In addition, since the expression of TDP-43 protein changes depending on the growth phase of cancer, model cells using cell lines derived from cancer cells are not suitable for screening for therapeutic and preventive drugs for neurodegenerative diseases.

Therefore, in the search for therapeutic agents and preventive agents for neurodegenerative diseases, neurodegenerative disease model cells that do not rely on the introduction of gene mutations and that can uniformly reproduce the pathology in the nerve cells of disease patients and the model cells were used. A screening method was eagerly desired.

Accordingly, the present invention provides a neurodegenerative disease model cell inducer, a neurodegenerative disease model cell preparation method, and a therapeutic or prophylactic agent for neurodegenerative disease that cause accumulation of insoluble TDP-43 protein in the nerve cell. It aims to provide a method.

As a result of intensive studies to solve the above problems, the present inventors have found an inducer having an inhibitory action on vitamin B1 uptake and causing accumulation of TDP-43 protein insolubilized in nerve cells. Was completed.

That is, the present invention provides an inducer of a neurodegenerative disease model cell that has an inhibitory action on vitamin B1 uptake and causes accumulation of insoluble TDP-43 protein in nerve cells.

The above-mentioned inducer is preferably amprolium or a pharmacologically acceptable salt thereof, or oxythiamine or a pharmacologically acceptable salt thereof.

The above-mentioned neurodegenerative disease model cell is preferably a model cell of frontotemporal lobar degeneration or amyotrophic lateral sclerosis.

The above-described nerve cell is preferably a non-cancerous nerve cell of a mammal.

The present invention also provides a method for producing a neurodegenerative disease model cell comprising a culturing step of culturing a mammalian non-cancerous nerve cell in the presence of the above inducer.

The above inducer is preferably 2 to 12 mmol / L of amprolium or a pharmacologically acceptable salt thereof.

In addition, the above culturing step is preferably a step of culturing mammalian non-cancerous neurons for 6 hours to 21 days.

The mammalian non-cancerous neuron is preferably a mammalian pluripotent stem cell-derived neuron, more preferably a human iPS cell-derived neuron, and a human normal iPS cell-derived neuron. More preferably, it is particularly preferably a human normal iPS cell-derived cholinergic neuron or a human normal iPS cell-derived choline acetyltransferase positive neuron.

Further, the present invention provides a contact step of bringing a test substance into contact with a neurodegenerative disease model cell prepared by the above preparation method, an accumulation amount of insoluble TDP-43 protein in the cell of the neurodegenerative disease model cell, An experimental group measurement step for measuring one or more evaluation items selected from the group consisting of the number of surviving degenerative disease model cells and the morphology of the neurodegenerative disease model cells, and an evaluation item measured in the experimental group measurement step as an index As described above, when the accumulated amount of the insolubilized TDP-43 protein is suppressed, when the survival number is decreased, and / or when the change in the form is suppressed, the test substance is used for treatment of neurodegenerative diseases. And a determination step (A) for determining that the substance is a drug and / or prophylactic drug candidate substance.

In addition, the present invention provides a contact step of bringing a test substance into contact with a neurodegenerative disease model cell prepared by the above preparation method, an accumulated amount of insoluble TDP-43 protein in the neurodegenerative disease model cell, the neurodegeneration An experimental group measurement step for measuring one or more evaluation items selected from the group consisting of the number of survival of the disease model cells and the morphology of the neurodegenerative disease model cells, and the neurodegenerative disease model cells not contacted with the test substance A control group measurement step for measuring one or more evaluation items selected from the group consisting of the accumulated amount of insoluble TDP-43 protein in the cell, the number of surviving neurodegenerative disease model cells and the morphology of the neurodegenerative disease model cells; The results of the evaluation items measured in the experimental group measurement step are insolubilized compared to the results of the evaluation items serving as the controls measured in the control group measurement step. When the accumulated amount of TDP-43 protein is suppressed, when the survival number is decreased, and / or when the change in the form is suppressed, the test substance is a therapeutic agent for neurodegenerative diseases and / or And a determination step (B) for determining that the substance is a prophylactic drug candidate substance.

The above form is preferably the length and / or density of neurites.

In the above screening method, the neurodegenerative disease is preferably FTLD or ALS.

According to the inducer of a neurodegenerative disease model cell that causes accumulation of TDP-43 protein insolubilized in nerve cells of the present invention, the pathology in nerve cells of patients with neurodegenerative diseases that does not depend on the introduction of gene mutations Can be uniformly reproduced, and a neurodegenerative disease model cell in which insoluble TDP-43 protein accumulates in the cell can be produced. Moreover, if the neurodegenerative disease model cell is used, a therapeutic or prophylactic agent for neurodegenerative disease can be screened.

An example of a procedure for preparing a neurodegenerative disease model cell in which insolubilized TDP-43 protein accumulates in the cell and a flow of screening for a therapeutic and / or prophylactic agent for neurodegenerative disease using the neurodegenerative disease model cell is shown. FIG. FIG. 2A is a Western blot image of expression of full-length TDP-43 and fragmented TDP-43 in the insoluble fraction of human normal iPS cell-derived cholinergic neurons treated with amprolium. FIG. 2B is a graph showing expression levels quantified from Western blot images of full-length TDP-43 and fragmented TDP-43 in an insoluble fraction of human normal iPS cell-derived cholinergic neurons treated with amprolium. FIG. 3A: Western blot image of expression of full-length TDP-43 and fragmented TDP-43 in the insoluble fraction of human normal iPS cell-derived cholinergic neurons treated with oxythiamine. FIG. 3B is a graph showing expression levels quantified from Western blot images of full-length TDP-43 and fragmented TDP-43 in an insoluble fraction of human normal iPS cell-derived cholinergic neurons treated with oxythiamine. FIG. 4A is a diagram showing an image analysis result of the number of surviving human normal iPS cell-derived cholinergic neurons by amprolium treatment. FIG. 4B is a diagram showing an image analysis result of neurite length of cholinergic neurons derived from human normal iPS cells by amprolium treatment. FIG. 4C is an example of images used for image analysis of human normal iPS cell-derived cholinergic neurons by solvent treatment and amprolium treatment. FIG. 5A is a graph showing the effect of existing ALS therapeutic agents (edaravone and riluzole) on the survival number of human normal iPS cell-derived cholinergic neurons by amprolium treatment. FIG. 5B shows the effect of existing ALS therapeutics (edaravone and riluzole) on the neurite length of human normal iPS cell-derived cholinergic neurons treated with amprolium.

Hereinafter, the present invention will be described in detail.

The inducer of a neurodegenerative disease model cell that causes accumulation of TDP-43 protein insolubilized in nerve cells of the present invention is characterized by having an inhibitory action on vitamin B1 uptake.

“Vitamin B1 uptake inhibitory action” means an action of inhibiting or suppressing vitamin B1 uptake into cells and reducing the amount of vitamin B1 in the cells. Examples of the substance having an inhibitory action on vitamin B1 uptake include amprolium or a pharmacologically acceptable salt thereof, oxythiamine or a pharmacologically acceptable salt thereof, or pyrithiamine. Pharmaceutically acceptable salt or oxythiamine or a pharmacologically acceptable salt thereof is preferable, and amprolium or a pharmacologically acceptable salt thereof is more preferable. Vitamin B1 uptake inhibitors such as amprolium or a pharmacologically acceptable salt thereof, oxythiamine or a pharmacologically acceptable salt thereof or pyrithiamine are commercially available.

Examples of “mammals” include humans, mice, rats, rabbits, dogs, cats, cows, horses, pigs, and monkeys, with humans being particularly preferred.

“Non-cancerous neuron” means a non-cancerous neuron. The nerve cell is preferably a cultured cell, and more preferably a nerve cell cultured in a state that retains properties close to those of a normal in vivo nerve cell.

Examples of “mammalian non-cancerous nerve cells” include, for example, mammalian primary cultured neurons, mammalian tissue stem cell-derived neurons, or mammalian pluripotent stem cell-derived neurons. It is preferably an animal pluripotent stem cell-derived neuron, more preferably a human pluripotent stem cell-derived neuron.

“A pluripotent stem cell” means a cell having both the ability to differentiate into all cells existing in a living body and the ability to self-proliferate. Examples of pluripotent stem cells include artificial pluripotent stem cells (hereinafter referred to as iPS cells), embryonic stem cells, reproductive stem cells, and Muse cells, and iPS cells are preferred.

“A pluripotent stem cell-derived neuron” means a neuron obtained by inducing differentiation from a pluripotent stem cell. The pluripotent stem cell-derived neuron is preferably an iPS cell-derived neuron, and more preferably a human iPS cell-derived neuron.

An “iPS cell” is a stem cell artificially prepared by introducing a specific reprogramming factor into a somatic cell in the form of DNA, mRNA or protein, and has properties similar to embryonic stem cells, such as various biological tissues. It is a cell having the ability to differentiate into cells and the ability to self-proliferate.

The human iPS cell-derived nerve cell is preferably a human normal iPS cell-derived nerve cell. The human normal iPS cell-derived neuron is preferably a human normal iPS cell-derived cholinergic neuron or a human normal iPS cell-derived choline acetyltransferase positive neuron.

Primary cultured neurons can be purchased from ATCC or Lonza, for example. Moreover, it is prepared by removing the brain of a mammalian fetus and isolating and culturing it by the method described in the literature (Kaech et al., Nature Protocols, 2006, Vol. 1, No. 5, p. 2406-2415). You can also.

Human normal iPS cell-derived cholinergic neuron or human normal iPS cell-derived choline acetyltransferase positive neuron, for example, after culturing human normal iPS cell in F12 / DMEM / N2 medium and inducing neural progenitor cell, A method of culturing for 20 days in the presence of retinoic acid and sonic hedgehog (Briscoe et al., Current Opinion in Neurobiology, 2001, Vol. 11, No. 1, p. 43-49), F12 / DMEM After culturing normal human iPS cells in / N2 medium and inducing neural progenitor cells, three transcription factors Ngn2, Isl-1 and Lhx3 were forced to be expressed in neural progenitor cells, and then retinoic acid and sonic hedgehog were also present By continuing the culture for 7 days under Seisuru method (Hester et al., Molecular Therapy, 2011 years, Vol. 19, No. 19, P.1905-1949) can be prepared in like. In addition, human normal iPS cell-derived choline acetyltransferase positive neurons can be purchased as cell kits from Reprocell in Japan.

“TDP-43 protein” is a heterogeneous nuclear riboprotein that is localized in the nucleus and binds to ribonucleic acid and other riboproteins to stabilize ribonucleic acid, alternative splicing, and / or transcriptional regulation. It is a protein involved in such processes. In addition to trans-activation response element DNA-binding protein of 43 kDa, TDP-43 protein is also known as transactive response DNA binding binding 43 kDa, transactivation response-43. It is also written as 43 kDa TAR DNA-binding protein. In the present specification, unless otherwise specified, “TDP-43 protein” includes all insolubilized TDP-43 protein, full-length TDP-43 protein, and fragmented TDP-43 protein.

The “insolubilized TDP-43 protein” means a protein that has been changed to a non-solubilized property under normal protein extraction conditions (for example, conditions using a surfactant, etc.) in which the TDP-43 protein is solubilized. . Examples of insolubilized TDP-43 protein include fragmented TDP-43 protein, abnormally phosphorylated TDP-43 protein, and ubiquitinated TDP-43 protein.

“Fragmented TDP-43 protein” means that the full-length TDP-43 protein has been broken down into smaller fragments by the action of proteolytic enzymes and the like. Although it will not specifically limit if it is small, For example, TDP-43 protein cut | disconnected by 35 kDa or 25 kDa is mentioned.

“Abnormally phosphorylated TDP-43 protein” means a protein in which a part of amino acid residues of TDP-43 protein is phosphorylated by a phosphorylase reaction, for example, a plurality of sites in the C-terminal region. Examples thereof include TDP-43 protein in which (Ser403, Ser404, Ser409, Ser410 and the like) are phosphorylated.

The “ubiquitinated TDP-43 protein” is a TDP-43 protein in which the ubiquitin protein is added by an isopeptide bond by the action of ubiquitin ligase or the like.

“Accumulation of insolubilized TDP-43 protein in nerve cells” means that the insolubilized TDP-43 protein increases in nerve cells. In particular, it is preferably in the cytoplasm of a nerve cell.

In addition, the present invention is characterized by providing a method for producing a neurodegenerative disease model cell comprising a culturing step of culturing a mammal's non-cancerous nerve cell in the presence of the above inducer.

“Neurodegenerative disease” is a general term for progressive diseases in which a specific type of nerve cell group gradually degenerates and causes cell death in the central nervous system.

The above-mentioned neurodegenerative disease model cell is preferably a neurodegenerative disease model cell in which insolubilized TDP-43 protein is accumulated in the cell.

“A neurodegenerative disease model cell in which insolubilized TDP-43 protein is accumulated in the cell” is a cell in which the insolubilized TDP-43 protein is accumulated in the cell (preferably in the cytoplasm). It is a cell that mimics the pathological features of neurodegenerative diseases that are known to accumulate insoluble TDP-43 protein at any stage of the pathological state. Therefore, the neurodegenerative disease is not particularly limited as long as accumulation of insolubilized TDP-43 protein in nerve cells is known to occur at any stage of the disease state. Examples of neurodegenerative diseases in which accumulation of insolubilized TDP-43 protein in nerve cells is known to occur at any stage of the disease state include FTLD and ALS, with ALS being particularly preferred. The above pathological features include accumulation of insolubilized TDP-43 protein in neurons, decrease in the survival number of neurons (cell death), and change in neuronal morphology (neurite abnormality). Include. Preferably, the change in the morphology of the nerve cell is a change in neurite length and / or density.

In the above method for producing a neurodegenerative disease model cell, the inducer is preferably 0.01 to 1000 mmol / L, more preferably 0.1 to 100 mmol / L. Further, the above-mentioned inducer is preferably 0.01 to 1000 mmol / L amprolium or a pharmacologically acceptable salt thereof, and 0.1 to 100 mmol / L amprolium or a pharmacologically acceptable salt thereof. More preferably 2 to 12 mmol / L amprolium or a pharmacologically acceptable salt thereof, and 6 to 12 mmol / L amprolium or a pharmacologically acceptable salt thereof. It is particularly preferred that The inducer is preferably 0.01 to 100 mmol / L oxythiamine or a pharmaceutically acceptable salt thereof, and 0.1 to 10 mmol / L oxythiamine or a pharmacologically salt thereof. An acceptable salt is more preferable, and 0.6 to 3 mmol / L oxythiamine or a pharmacologically acceptable salt thereof is more preferable. The above inducer is preferably added and dissolved in, for example, a cell culture medium. Various buffer solutions (for example, HEPES buffer solution, phosphate buffer solution, phosphate buffer physiology) are contained in the cell culture medium. Saline, Tris-HCl buffer, borate buffer, acetate buffer, etc.).

The culture step is preferably a step of culturing for 6 hours to 21 days, more preferably a step of culturing for 1 to 14 days, further preferably a step of culturing for 3 to 14 days. The step of culturing for 7 days is particularly preferable.

The above-described method for producing a neurodegenerative disease model cell is obtained by treating mammalian non-cancerous nerve cells with 0.1 to 100 mmol / L amprolium or a pharmacologically acceptable salt thereof or 0.1 to 10 mmol / L. Preferably culturing for 1 to 14 days in the presence of oxythiamine or a pharmacologically acceptable salt thereof, wherein the mammalian non-cancerous nerve cells are treated with 6 to 12 mmol / L of amprolium or its It is more preferable to provide a step of culturing for 3 to 7 days in the presence of a pharmacologically acceptable salt or 0.6 to 3 mmol / L oxythiamine or a pharmacologically acceptable salt thereof. Further, the above-mentioned method for producing a neurodegenerative disease model cell comprises a method wherein human normal iPS cell-derived nerve cells are mixed with 0.1 to 100 mmol / L amprolium or a pharmacologically acceptable salt thereof or 0.1 to 10 mmol / L. More preferably, the method further comprises a step of culturing for 1 to 14 days in the presence of L oxythiamine or a pharmaceutically acceptable salt thereof. It is particularly preferable to provide a step of culturing for 3 to 7 days in the presence of a pharmacologically acceptable salt or 0.6 to 3 mmol / L oxythiamine or a pharmacologically acceptable salt thereof.

The present invention is also characterized by providing a screening method for therapeutic and / or prophylactic agents for neurodegenerative diseases using the neurodegenerative disease model cells prepared by the above method. The screening method includes a step of contacting a test substance with a neurodegenerative disease model cell prepared by the above method, an accumulation amount of insolubilized TDP-43 protein in the neurodegenerative disease model cell, the neurodegenerative disease model The experimental group measurement step of measuring one or more evaluation items selected from the group consisting of the number of cells surviving and the form of the neurodegenerative disease model cell, and the evaluation items measured in the experimental group measurement step as indicators, When the accumulated amount of insolubilized TDP-43 protein is suppressed, when the number of surviving cells is decreased, and / or when the change in morphology is suppressed, the test substance is a therapeutic agent for neurodegenerative diseases and / or Or the determination process (A) determined with it being a candidate substance of a preventive drug is provided.

More specifically, the screening method preferably includes the following steps (1) to (5).
(1) A neurodegenerative disease model cell production step comprising the above method of producing a neurodegenerative disease model cell;
(2) a contact step of bringing a test substance into contact with a neurodegenerative disease model cell;
(3) One or more evaluations selected from the group consisting of the accumulation amount of insoluble TDP-43 protein in the neurodegenerative disease model cell, the number of survival of the neurodegenerative disease model cell, and the morphology of the neurodegenerative disease model cell An experimental group measurement process for measuring items;
(4) A group consisting of the accumulated amount of insolubilized TDP-43 protein in the neurodegenerative disease model cells not contacted with the test substance, the number of surviving neurodegenerative disease model cells, and the form of the neurodegenerative disease model cells A control group measurement step for measuring one or more evaluation items selected from
(5) In the case where the accumulated amount of the insolubilized TDP-43 protein is suppressed in comparison with the result of the target evaluation item measured in the control group measurement step The step of determining that the test substance is a candidate substance for a therapeutic and / or prophylactic drug for neurodegenerative diseases when the number of survivors decreases and / or when the change in the form is suppressed (B ).

The screening method includes a step of measuring one or more evaluation items selected from the group consisting of the amount of insoluble TDP-43 protein in a neurodegenerative disease model cell, the number of viable cells, and the cell morphology. However, as the above evaluation items, the survival number and / or morphology of neurodegenerative disease model cells can be easily measured, and it is more preferable to measure both the survival number and morphology of neurodegenerative disease model cells. preferable.

The “test substance” is not limited to, but is not limited to, an in-vivo substance, a genetically modified substance, a natural product, or a chemically synthesized compound. Moreover, said test substance can be added after dissolving or suspending in a solvent. The solvent can be any suitable pharmaceutically acceptable carrier. Moreover, the addition amount, the number of additions, and the addition period of the test substance can be appropriately set according to the type of cells, the type of test substance, and the like. Moreover, the therapeutic agent or preventive agent of the known neurodegenerative disease for comparing efficacy may be sufficient.

The contact between the test substance and the above-mentioned neurodegenerative disease model cell is, for example, a cell culture medium or various buffer solutions (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, The test substance can be added to an acid buffer, an acetate buffer, etc.), and the cells can be incubated for a certain period of time. What is necessary is just to set suitably the addition period, timing, frequency | count, and density | concentration of said test substance by the efficacy of a test substance. For example, the timing of adding the above-mentioned test substance may be added before, simultaneously with, or after adding the above inducer (preferably, amprolium, oxythiamine or a pharmacologically acceptable salt thereof). Preferably, it is simultaneous addition with said inducer.

“Neurodegenerative disease model cell not contacted with test substance” means a corresponding neurodegenerative disease model cell to which the test substance is not added, and the neurodegenerative disease model cell is referred to as “contacted with test substance” It may be a different cell from the “neurodegenerative disease model cell” or the same cell. For example, in the case of the same cell, the neurodegenerative disease model cell before adding the test substance corresponds to “a neurodegenerative disease model cell not contacted with the test substance”, and the neurodegenerative disease after adding the test substance The model cell corresponds to “a neurodegenerative disease model cell contacted with a test substance”. Since the neurodegenerative disease model cell of the present invention is a neurodegenerative disease model cell capable of uniformly reproducing the pathology in the neuronal cell of a neurodegenerative disease patient (preferably, FTLD and ALS), it is a nerve not contacted with a test substance. As the degenerative disease model cell, it is preferable to use a cell that is cultured under the same conditions, using a different cell from the neurodegenerative disease model cell contacted with the test substance. “No test substance contacted” means that the same amount of solvent (blank) as the test substance is added instead of the test substance or a negative control substance that does not affect the measurement item is added. included.

The experiment group measurement step and the control group measurement step may be performed at the same time or at different times. For example, the amount of insoluble TDP-43 protein in the cell of the neurodegenerative disease model cell not contacted with the test substance as a control, the number of surviving neurodegenerative disease model cells and / or the form of the neurodegenerative disease model cells A value measured in advance may be used. The experiment group measurement step and the control group measurement step are preferably performed at the same time.

The method for measuring the insolubilized TDP-43 protein amount may be any method as long as the amount of TDP-43 protein can be quantitatively detected. For example, a method may be mentioned in which a protein solution of an insoluble fraction is prepared from cells, and TDP-43 protein in the protein solution of the insoluble fraction is detected by an immunological technique. Alternatively, the cells may be directly immunostained to detect the amount of cytoplasmic inclusions where the insolubilized TDP-43 protein is localized. A method of detecting TDP-43 protein in the protein solution of the insoluble fraction by an immunological technique is preferred.

In the method of detecting the TDP-43 protein in the protein solution of the insoluble fraction by immunological techniques, the protein solution of the insoluble fraction from the cell may be prepared immediately after the collection of the cell, or the protein of the cell May be carried out at a later date after being properly stored (for example, after cryopreservation) under conditions that are not degraded or denatured. In order to detect the TDP-43 protein in the protein solution of the insoluble fraction by an immunological technique, it is preferable to perform a pretreatment for extracting the protein from the collected cells. The protein can be extracted by, for example, a tissue disruption method or a method using a protein extraction buffer. Specifically, after adding a buffer that dissolves only the soluble fraction in the cells, the cells are disrupted by ultrasonic waves, centrifuged, and then the supernatant is recovered to extract the protein of the soluble fraction. The precipitate after the centrifugation is further suspended using a buffer capable of dissolving the insoluble fraction, and the protein of the insoluble fraction can be extracted by sonication again (see Example 1). Protein extraction may be performed immediately after cell recovery, or may be extracted at a later date after appropriate storage (eg, after cryopreservation) under conditions where the protein is not degraded or denatured. The protein extract may be appropriately stored (for example, cryopreserved) under conditions where the protein is not decomposed or denatured until the next operation.

The immunological technique for measuring the amount of TDP-43 protein in the protein solution of the insoluble fraction is not particularly limited, but an immunological technique for detection with an anti-TDP-43 antibody is preferred. For example, it can be measured using Western blot, dot blot, immunoprecipitation, RIA, protein array, ELISA, etc., but using anti-TDP-43 antibody, full-length TDP-43 protein and fragmented TDP-43 Measurement by Western blotting in which protein is detected as a band is preferred.

The method for measuring the amount of TDP-43 protein insolubilized by Western blotting is a known method, and is not particularly limited. For example, the same amount of the above protein extract is applied to a well of a polyacrylamide gel, and SDS- After electrophoresis by PAGE, it is transferred to a PVDF membrane, and the TDP-43 protein transferred on the membrane is used with an anti-TDP-43 antibody and a labeled secondary antibody that recognizes the anti-TDP-43 antibody. There is a method of detecting as a specific band and quantifying the band with commercially available image analysis software.

The anti-TDP-43 antibody may be any antibody that can detect the TDP-43 protein, and may be a monoclonal antibody or a polyclonal antibody. In some cases, an antibody fragment, for example, Fab ', Fab, F (ab') 2 can be used. Moreover, these antibodies can be produced by a known method.

The method for measuring the number of surviving neurodegenerative disease model cells is not particularly limited, but can be measured using a known method. For example, absorbance measurement using MTT method, WST-1 method, WST-8 method or Almer Blue method, staining using PI (propidium iodide), counting using a flow cytometer, microscope Examples include a method of measuring the number of cell nuclei by analyzing visual measurements or capturing captured images below, or a method of measuring the number of cells by identifying only nerve cells from the coexistence of nerve-like cell morphology and nuclear morphology, etc. However, a method of identifying only nerve cells from the coexistence of nerve-like cell morphology and nuclear morphology and measuring the number of cells is preferred.

The method for identifying only nerve cells from the coexistence of neuronal cell morphology and nuclear morphology and measuring the number of cells is not particularly limited. For example, the neuronal cell morphology and nuclear morphology coexist under a microscope. A method of visually measuring cells or nuclear staining with a nuclear-specific marker and cell labeling with a neuron-specific marker, measuring the overlapping portion between the nuclear staining and the cell label, and measuring the nerve cell (having the overlapping portion) A method in which cells are the main body of nerve cells). Nuclear staining and cell labeling may be observed visually under a microscope, or an image taken with a cell image acquisition device may be analyzed with image analysis software, but an image taken with a cell image acquisition device may be analyzed. It is preferable to analyze by.

The cell labeling method is not particularly limited, and a known method can be used. For example, a method of labeling by immunostaining using an antibody that binds to a neuron-specific marker protein, or a reporter gene that is expressed by introducing a vector linking a reporter gene downstream of the promoter region of a protein that is specifically expressed in a neuron into the cell. There is a method of detecting a protein as a label. The above-described cell labeling method is preferably a method of labeling by immunostaining using an antibody that binds to a nerve cell-specific marker protein.

The above immunostaining can be performed by a known method. For example, a method in which a primary antibody that binds to a neuron-specific marker is directly labeled with a labeling substance is added to a cell sample and immunostaining is performed, or an unlabeled primary antibody and the primary antibody specifically A method of performing immunostaining using a secondary antibody that is recognized and labeled with a labeling substance is exemplified. An unlabeled primary antibody and a labeled substance that specifically recognizes the primary antibody are labeled. A method of performing immunostaining using a secondary antibody is preferred (see Examples 1 and 2 below).

The primary antibody that binds to the above neuron-specific marker is not particularly limited, and examples thereof include anti-βIII-tubulin antibody, anti-NeuN antibody, anti-MAP2 antibody, and anti-Neurofilament antibody. As a primary antibody that binds to a neuron-specific marker, an anti-βIII-tubulin antibody is preferable. These antibodies may be monoclonal antibodies or polyclonal antibodies.

The secondary antibody labeled with a label substance that specifically recognizes the primary antibody may be a monoclonal antibody or a polyclonal antibody.

The above-mentioned label substance is not particularly limited, and examples of the fluorescent label substance include FITC, TEXAS RED, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 568, CY3 or CY5. Moreover, Horse radish peroxidase, Alkaline phosphatase, etc. can be used as an enzyme label substance. As the label substance, a fluorescent label substance is preferable.

The method for nuclear staining is not particularly limited as long as it is a method that can be distinguished from the color of immunostaining of a neuron-specific marker. Examples of fluorescent nuclear staining include staining methods using DAPI, PI, or Hoechst. . Examples of nuclear staining for bright field observation include staining methods such as hematoxylin staining or methyl green staining. As the above-mentioned nuclear staining method, fluorescent nuclear staining is preferable.

Measurement items of “morphology of neurodegenerative disease model cell” include, for example, neurite length, degree of neurite fragmentation, neurite count, neurite density, neurite swelling degree, or neurite bead The length of the neurite and / or the density are preferable. The density of neurites may be measured as the product of fluorescence intensity and area on the neurite image.

The method for measuring the morphology of the above-mentioned neurodegenerative disease model cell is not particularly limited. The method of analyzing by the image analysis software is preferable.

The cell image acquisition device is not particularly limited, and examples thereof include ToxInsight, CellInsight, IN Cell Analyzer, Image Xpress, Opera, and Operata.

The above-described image analysis software is not particularly limited, and examples thereof include Cellomics Scan, In Cell Investigator, Meta Xpress, Harmony, Image J, or NIH Image.

“Changes in the morphology of neurodegenerative disease model cells” include shortening of neurites, fragmentation of neurites, decrease in the number of neurites, decrease in neurite density, swelling of neurites, and changes in neurite beads Although mentioned, shortening of neurites and reduction of density are preferred. “Suppression of morphological change” means, for example, that the degree of shortening of neurites is small compared to neurodegenerative disease model cells not contacted with the above test substance, neurite fragmentation is small, neurite It means that the decrease in the number is suppressed, the decrease in the density of neurites is suppressed, the swelling of neurites is suppressed, or the bead-like change of neurites is suppressed.

The method for screening a therapeutic agent or preventive agent for a neurodegenerative disease described above is a therapeutic agent for a neurodegenerative disease, which is known to cause accumulation of insolubilized TDP-43 protein in nerve cells at any stage of the disease state. A screening method for prophylactic drugs is preferred. Examples of neurodegenerative diseases in which accumulation of insolubilized TDP-43 protein in nerve cells is known to occur at any stage of the disease state include FTLD and ALS, with ALS being particularly preferred.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Example 1) Changes in nerve cells by treatment with an inducer having an inhibitory action on vitamin B1 uptake Neuronal differentiation ReproNeuro Ach (ReproCell, RCESDA001) was used as a cholinergic neuron derived from normal human iPS cells.

The coating plate was produced according to the protocol of ReproCell. That is, 0.02% poly-L-lysine solution (0.1% solution was diluted 5 times with PBS) was added to a polystyrene 96-well plate or 12-well plate and incubated at 37 ° C. for 2 hours or more. After removing the Poly-L-lysine solution from the plate, the plate was washed twice with PBS, then the coating solution (Repro Coat) was added to the plate, incubated overnight at 37 ° C., and used within one week. The coating solution was removed before cell seeding.

Cell seeding was performed according to the protocol of ReproCell. That is, the cell vial was taken out of dry ice or liquid nitrogen, immediately heated for 90 seconds in a 37 ° C. water bath, then transferred to Whating Medium, and centrifuged at 350 × g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in a medium (added with Culture medium, ACh Additive, and Penicillin / Streptomycin) and seeded on the coating plate. The seeding date was day 0, and thereafter, medium was changed by half each for day 3 and day 7, and day 14 cells in which sufficient extension of neurites was observed were used in the following experiments.

2. Cell treatment with an inducer having an inhibitory action on vitamin B1 uptake The cell culture supernatant is removed, and an inducer with an inhibitory action on vitamin B1 uptake is used as amprolium (6.0 mmol / L or 12 mmol / L for 12-well plates, 96-well plates). 2-12 mmol / L) or fresh medium containing oxythiamine (final concentration: 3 mmol / L or 0.6 mmol / L) was added to the cells (referred to as “Amprolium-treated group” and “Oxythiamine-treated group”, respectively) ). As a control, only solvent (0.1% DMSO) was added (no vitamin B1 uptake inhibitor). Fresh medium was added to the cells (this is referred to as the “solvent treatment group”). In the case of a 12-well plate, the cells were further cultured for 3 days. In the case of a 96-well plate, the cells were further cultured for 7 days (after 4 days from the start of cell treatment, half of the old medium was removed and half of the new medium containing each additive was added).

3. Insolubilization and fragmentation of TDP-43 (cell protein fraction and Western blot)
The culture supernatant is removed from the cells cultured for 3 days in a 12-well plate, and the cells are washed with ice-cold PBS, followed by addition of RIPA Buffer mixture (including Protease inhibitor cocktail, Pho Stop and UREA buffer), and ice-cooled. Then, the mixture was allowed to stand for 30 minutes, and then sonicated to recover the cell disruption solution. The amount of protein in the cell lysate at this point was quantified by the BCA method. The cell lysate was centrifuged at 100,000 × g at 4 ° C., and the supernatant was recovered as a soluble fraction. Furthermore, the pellet was similarly centrifuged twice with RIPA Buffer mixture (4 ° C, 100,000 xg), and UREA buffer was added to the resulting pellet, and the lysate dissolved was recovered as an insoluble fraction. did.

The recovered insoluble fraction was subjected to electrophoresis by SDS-PAGE and then transferred from the gel to the PVDF membrane. The PVDF membrane is immersed in a blocking solution (Blocking One, Nacalai Tesque), blocked at room temperature for 30 minutes, and then incubated overnight at 4 ° C. with a rabbit anti-TDP-43 antibody (Protein Tech, diluted 1000 times with an antibody diluent). And reacted. Next, the PVDF membrane was washed 3 times with TBST and then reacted with HRP-labeled rabbit IgG antibody (GE Healthcare, diluted 10000 times with antibody diluent) at room temperature for 1 hour. Furthermore, after washing the PVDF membrane 3 times with TBST, ECL Prime (GE Healthcare) was added to the PVDF membrane and allowed to stand for 5 minutes, and then the luminescence detection device (Chemi-Doc XRS Plus, Bio-Rad) was installed. Was used to detect luminescence. The signal intensity of each band was detected from the obtained image using image analysis software Image Lab (BIO-RAD), and the relative expression level of TDP-43 protein was measured.

4). Measurement of neuronal survival and morphology (immunocytochemistry)
The culture supernatant was removed from the cells cultured in a 96-well plate for 7 days, the cells were washed twice with PBS, 4% PFA was added, and the mixture was fixed at room temperature for 30 minutes. The cells were washed twice with PBS, then PBS containing 0.1% Triton X-100 was added, and permeabilization was performed at room temperature for 30 minutes. After washing the cells twice with PBS, blocking solution (Blockng One, Nacalai Tesque) was added and blocked at room temperature for 30 minutes, then mouse anti-βIII-tubulin antibody (diluted with blocking solution) and 2 hours at room temperature. Alternatively, the reaction was allowed to incubate overnight at 4 ° C. The cells were washed twice with PBS, and reacted with Alexa Fluor 488-labeled anti-mouse IgG antibody (diluted with a blocking solution) for 1 hour at room temperature under light-shielded conditions. The cells were washed twice with PBS, and then DAPI (diluted with PBS) was added to the cells and reacted at room temperature for 10 minutes for nuclear staining. Finally, 100 μL / well of PBS was added and the cells were photographed using ToxInsight.

Quantitative analysis of the survival number of neurites and the length of neurites was performed using images taken with the imaging device ToxInsight (ThermoFisher Scientific), and the neurite analysis protocol DeveNopenteTP of image analysis software ArrayScan (ThermoFisherScientific). The analysis was performed using v4.1. Data analysis was performed using Cellomics (ThermoFisher scientific) and Microsoft Excel.

In Example 1 above. ~ 4. The flow is shown in FIG.

5. Results The measurement results of the expression levels of full-length TDP-43 protein and fragmented TDP-43 protein in the insoluble fraction are shown in FIG.

Fig. 2 shows the results of amprolium treatment. FIG. 2A shows a Western blot image. The vertical axis of the graph in FIG. 2B shows the expression level of TDP-43 protein in the insoluble fraction, and the bar graph shows full-length TDP-43 protein (43 kDa, black), fragmented TDP-43 protein (35 kDa is light gray, 25 kDa). Is the dark gray). The horizontal axis shows the solvent treatment group (“solvent” in the figure), the amprolium 6.0 mmol / L treatment group, and the amprolium 12 mmol / L treatment group from the left.

FIG. 3 shows the results of oxythiamine treatment. FIG. 3A shows a Western blot image. The vertical axis of the graph in FIG. 3B shows the expression level of TDP-43 protein in the insoluble fraction, and the bar graph shows full-length TDP-43 protein (43 kDa, black), fragmented TDP-43 protein (35 kDa is light gray, 25 kDa). Is the dark gray). The horizontal axis shows, from the left, a solvent treatment group (“solvent” in the figure), an oxythiamine 3.0 mmol / L treatment group, and an oxythiamine 0.6 mmol / L treatment group.

In the cell treatment with amprolium 6.0 mmol / L or 12 mmol / L, the full length TDP-43 protein (43 kDa) and the fragmented TDP-43 protein (25 kDa and 35 kDa) in the insoluble fraction compared to the control solvent treatment group. The expression level increased (FIG. 2B). Even when oxythiamine 3.0 mmol / L or 0.6 mmol / L was treated, the full-length TDP-43 protein (43 kDa) and the fragmented TDP-43 protein (25 kDa and 35 kDa) in the insoluble fraction compared to the solvent-treated group. ) Increased (FIG. 3B). From these results, it was shown that the amount of full-length TDP-43 protein and fragmented TDP-43 protein in the insoluble fraction increased in human normal iPS cell-derived cholinergic neurons by treatment with amprolium or oxythiamine.

Fig. 4 shows the measurement results of the survival number and morphology of nerve cells.

The vertical axis of FIG. 4A indicates the survival number of nerve cells (“number of nerve cells” in the figure), and the horizontal axis indicates the concentration of amprolium. The vertical axis of FIG. 4B indicates the total neurite length / number of neurons, that is, the length of neurite per nerve cell, and the horizontal axis indicates the concentration of amprolium. FIG. 4C shows immunostained images with anti-βIII-tubulin when nerve cells were treated with a solvent or with 8 mmol / L of amprolium.

In the cell treatment with amprolium 6 mmol / L, the survival number of cholinergic neurons derived from human normal iPS cells was reduced by 45%, and the neurite length per nerve cell was reduced by 25%. In addition, cell treatment with amprolium 8 mmol / L reduced the survival number of cholinergic neurons derived from human normal iPS cells by 59%, and the neurite length per nerve cell decreased by 36%. This result shows that in human normal iPS cell-derived cholinergic neurons, the higher the concentration of amprolium, the smaller the number of surviving neurons and the shorter the neurite length per nerve cell. It was.

Thus, it has been shown that amprolium or oxythiamine causes accumulation of insolubilized TDP-43 protein in the cells of neurons, and further decreases the survival number of neurons and shortens neurites of neurons. .

From this result, an inducer having an inhibitory action on vitamin B1 uptake causes accumulation of insoluble TDP-43 protein in the cells of non-cancerous nerve cells of mammals. Furthermore, it is a pathological feature of neurodegenerative diseases. It has been shown to cause a decrease in the survival number of certain neurons and shortening of neurites of neurons.

(Example 2) Effects of existing therapeutic agents on nerve cells treated with an inducer having an inhibitory action on vitamin B1 uptake Neuronal differentiation ReproNeuro Ach (ReproCell, RCESDA001) was used as a cholinergic neuron derived from normal human iPS cells.

The coating plate was produced according to the protocol of ReproCell. Specifically, 0.02% poly-L-lysine solution (0.1% solution diluted 5 times with PBS) was added to a 96-well plate made of polystyrene, and incubated at 37 ° C. for 2 hours or more. After removing the Poly-L-lysine solution from the plate, the plate was washed twice with PBS, then a coating solution (Repro Coat) was added, incubated overnight at 37 ° C., and used within one week. The coating solution was removed before cell seeding.

Cell seeding was performed according to the protocol of ReproCell. That is, the cell vial was taken out of dry ice or liquid nitrogen, immediately heated for 90 seconds in a 37 ° C. water bath, then transferred to Whating Medium, and centrifuged at 350 × g for 5 minutes at room temperature. After centrifugation, the supernatant was removed, and the cells were suspended in a medium (added with Culture medium, ACh Additive, and Penicillin / Streptomycin) and immediately seeded on the coating plate. The seeding date was day 0, and thereafter, medium was changed by half each for day 3 and day 7, and day 14 cells in which sufficient extension of neurites was observed were used in the following experiments.

2. Treatment of cells with an inducer having an inhibitory action on vitamin B1 uptake and treatment of existing therapeutic agents The culture supernatant of the cells is removed, and amprolium (final concentration: 7 mmol / L) which is an inducer having an inhibitory action on vitamin B1 uptake is used. Fresh media containing ALS therapeutic (edaravone or riluzole) was added to the cells. They are called the edaravone treatment group and the riluzole treatment group, respectively. As a comparison, cells added with a new medium containing only solvent (0.1% DMSO) (not containing amprolium and existing ALS therapeutic agent) (referred to as a solvent treatment group) and amprolium (final concentration: 7 mmol / L) only (not containing the existing ALS therapeutic agent) was added to cells supplemented with a new medium (referred to as a control group). After adding the medium, the cells were further cultured for 7 days.

3. Measurement of neuronal survival and morphology (immunocytochemistry)
After culturing for 7 days, the culture supernatant was removed, the cells were washed twice with PBS, 4% PFA was added, and the mixture was fixed at room temperature for 30 minutes. After the cells were washed twice with PBS, PBS containing 0.1% Triton X-100 was added and permeabilized for 20 minutes at room temperature. After washing the cells twice with PBS, blocking solution (5-fold diluted Blockone, Nacalai Tesque) was added and blocked at room temperature for 40 minutes, and then mouse anti-βIII-tubulin antibody (diluted with blocking solution) was added at room temperature. The reaction was incubated for 2 hours or overnight at 4 ° C. The cells were washed twice with PBS, and reacted with Alexa Fluor 555-labeled anti-mouse IgG antibody (diluted with a blocking solution) for 1 hour at room temperature under light-shielded conditions. The cells were washed twice with PBS, and then DAPI (diluted with PBS) was added to the cells and reacted at room temperature for 10 minutes for nuclear staining. Finally, 200 μL / well of PBS was added and the cells were photographed using IN Cell Analyzer 2200 (GE Healthcare).

Quantitative analysis of the survival number of nerve cells and the length of neurites was performed by analyzing the captured images using image analysis software IN Cell Investigator (GE Healthcare). Statistical analysis used t-test. As a result of statistical analysis, when the P value was less than 0.05, it was judged to be statistically significant.

4). Results The results are shown in FIG.

The vertical axis of FIG. 5A indicates the number of surviving nerve cells (in the figure, “number of nerve cells”), and the horizontal axis indicates amprolium and the test substance (existing ALS therapeutic agent) and their concentrations. From left to right, a solvent treatment group treated with only 0.1% DMSO (“solvent” in the figure), a control group treated with only amprolium (7 mmol / L) (“no test substance” in the figure), amprolium ( 7 mmol / L) and a group treated with edaravone (1 and 10 μmol / L) as a test substance, and a group treated with amprolium (7 mmol / L) and riluzole (1 and 3 μmol / L) as a test substance. The vertical axis of FIG. 5B shows the total neurite length / number of neurons, that is, the length of neurite per nerve cell, and the horizontal axis shows amprolium and test substance (existing ALS therapeutic agent) and The concentration is shown. The symbol * (asterisk) in the figure indicates that the difference between the control group (“no test substance” in the figure) and the result (value on the vertical axis) is statistically significant (P <0.05). ).

Nerve cells cultured with the addition of edaravone (10 μmol / L) or riluzole (3 μmol / L) simultaneously with amprolium showed a decrease in the number of surviving neurons compared to the control group treated with amprolium alone. Was significantly suppressed (FIG. 5A).

On the other hand, the effects of edaravone or riluzole on neurite shortening caused by amprolium were only weakly suppressed (FIG. 5B). Edaravone and riluzole have been approved and used as ALS therapeutics, but their effects are both known to be limited, such as a life-prolonging effect of several months or a survival-prolonging effect without an event. The lack of a sufficient inhibitory effect on the shortening of neurites caused by amprolium is thought to reflect the limited clinical effects of edaravone and riluzole. Therefore, it was shown that the nerve cells treated with amprolium are a model that well reflects the pathologic characteristics of ALS, and at the same time, a model that can accurately evaluate the effect on drugs.

From the above results, it was clarified whether the neurodegenerative disease model cells prepared using the above inducer can be used for screening for therapeutic or prophylactic agents for neurodegenerative diseases.

According to the inducer that causes accumulation of TDP-43 protein insolubilized in nerve cells of the present invention, it is possible not to depend on the introduction of gene mutation and to uniformly reproduce the pathology in nerve cells of patients with neurodegenerative diseases. A neurodegenerative disease model cell in which insolubilized TDP-43 protein accumulates in the cell can be prepared. Moreover, if the neurodegenerative disease model cell is used, a therapeutic or prophylactic agent for neurodegenerative disease can be screened.

Claims (11)

1. An inducer of a neurodegenerative disease model cell having an inhibitory action on vitamin B1 uptake and causing accumulation of a trans-activity-responsive DNA-binding protein-43 insolubilized in nerve cells.
2. The inducer according to claim 1, which is amprolium or oxythiamine or a pharmacologically acceptable salt thereof.
3. The inducer according to claim 1 or 2, which is a model cell for frontotemporal lobar degeneration or amyotrophic lateral sclerosis.
4. A method for producing a neurodegenerative disease model cell, comprising a culture step of culturing a non-cancerous nerve cell of a mammal in the presence of the inducer according to any one of claims 1 to 3.
5. The preparation method according to claim 4, wherein the inducer is 2 to 12 mmol / L amprolium or a pharmacologically acceptable salt thereof.
6. The production method according to claim 4 or 5, wherein the culturing step is a step of culturing mammalian non-cancerous neurons for 6 hours to 21 days.
7. The production method according to any one of claims 4 to 6, wherein the nerve cell is a human induced pluripotent stem cell-derived nerve cell.
8. Contacting a test substance with a neurodegenerative disease model cell produced by the production method according to any one of claims 4 to 7,
   One or more selected from the group consisting of the accumulated amount of insolubilized transactivity-responsive DNA binding protein-43 in the neurodegenerative disease model cell, the number of survival of the neurodegenerative disease model cell, and the form of the neurodegenerative disease model cell An experimental group measurement process for measuring evaluation items;
   Using the evaluation item measured in the experimental group measurement step as an index, when the accumulated amount of the insolubilized transactivity-responsive DNA-binding protein-43 is suppressed, when the number of survivors decreases, and / or when the morphology changes Determination step (A) for determining that the test substance is a candidate substance for a therapeutic and / or prophylactic drug for neurodegenerative diseases,
   A screening method for a therapeutic or prophylactic agent for neurodegenerative diseases.
9. Contacting a test substance with a neurodegenerative disease model cell produced by the production method according to any one of claims 4 to 7,
   One or more selected from the group consisting of the accumulated amount of insolubilized transactivity-responsive DNA binding protein-43 in the neurodegenerative disease model cell, the number of survival of the neurodegenerative disease model cell, and the form of the neurodegenerative disease model cell An experimental group measurement process for measuring evaluation items;
   The accumulation amount of insolubilized transactivity-responsive DNA-binding protein-43 in the neurodegenerative disease model cell not contacted with the test substance, the number of survival of the neurodegenerative disease model cell, and the form of the neurodegenerative disease model cell A control group measurement step for measuring one or more evaluation items selected from the group;
   The result of the evaluation item measured in the experimental group measurement step is more than the result of the control evaluation item measured in the control group measurement step. When the number of survivors is reduced, and / or when the morphological change is suppressed, the test substance is a candidate for a therapeutic and / or preventive drug for neurodegenerative diseases. A determination step (B) for determining;
   A screening method for a therapeutic or prophylactic agent for neurodegenerative diseases.
10. 10. The screening method according to claim 8 or 9, wherein the form is a neurite length and / or density.
11. The screening method according to any one of claims 8 to 10, wherein the neurodegenerative disease is frontotemporal lobar degeneration or amyotrophic lateral sclerosis.
    

Images