Method for inducing synapse formation in nerve cells and microbeads used in the method.

Abstract

The purpose of the present invention is to provide a method for inducing a presynapse formation in human neurons.　According to the present invention, provided are: a method for inducing a presynapse formation in human neurons, the method comprising co-culturing the peripheral neurons with at least one LRRTM (Leucine-rich repeat transmembrane neuronal protein) molecule selected from the group consisting of LRRTM family molecules or microbeads of which a fusion protein including the same is immobilized on the surface (here, the LRRTM molecules or the fusion protein including the molecules are immobilized via a linker on the surface of the microbeads); and microbeads used in said method.

Description

Technical Field

[0001] The present invention relates to a method for inducing the formation of the presynaptic part of synapses of nerve cells. The present invention also relates to microbeads used in the method.

Background Art

[0002] Central nerve cells and peripheral nerve cells, which are nerve cells, exchange signals between cells via synapses and control human emotions and behaviors. A synapse is a gap formed between an axon, which is the presynaptic part, and a dendritic process (central nervous system) or a target cell such as skeletal muscle or an organ (peripheral nervous system), which is the postsynaptic part, and a chemical substance released from the presynaptic part binds to a receptor present in the postsynaptic part to transmit a signal. Synapse formation is triggered by the interaction of specific membrane proteins expressed in the presynaptic and postsynaptic parts, which is then converted into an intracellular signal. Subsequently, proteins involved in the release of chemical substances accumulate in the presynaptic part, and proteins that receive them and transmit them downstream accumulate in the postsynaptic part to form a special structure, thereby completing synapse formation.

[0003] There are multiple membrane ligands and their receptors related to the initiation of synapse formation in the central nervous system, and since the cell types and synapse types (excitatory synapses or inhibitory synapses) that act through their combinations are different (Non-Patent Document 1), the mechanisms of temporal and spatial control of specific synapse formation in specific cells are mostly unknown. Furthermore, most of the previous findings on synapse formation mainly used primary cultures of rodent hippocampal neurons and cerebral cortical neurons (Non-Patent Documents 2 and 3), and it is unclear whether their mechanisms are common to humans.

[0004] Regarding peripheral nervous system synapse formation, it has only been reported in mice that a membrane protein Lrp4 (LDL-receptor related protein 4) expressed in skeletal muscle, which is the postsynaptic part, induces synapse formation via an unknown receptor expressed in motor neurons (Non-Patent Document 4), but most of it remains unclear.

[0005] The LRRTM family is a family of synaptic organizer molecules on the postsynaptic terminal side of the central nervous system, and four types, LRRTM1 to LRRTM4, have been reported in humans and mice. LRRTM2 (Leucine-rich repeat transmembrane protein 2) is a membrane-bound protein expressed in the postsynaptic terminal and has been reported to induce the formation of excitatory presynaptic terminals in primary cultured hippocampal neurons of mice (Non-Patent Literature 5). However, it is unclear whether LRRTM2 also induces presynaptic terminal formation in human central nervous system cells. Furthermore, it has been reported that LRRTM2 induces excitatory synapses in mouse hippocampal neurons, similar to neuroligin 1 (Non-Patent Literature 6), and in that report, the LRRTM2 ligand is reported to be neurexin, similar to neuroligin 1.

[0006] To date, there have been no reports on factors that induce synapse formation in human nerve cells, nor have there been any reports on methods for inducing presynaptic formation. To elucidate human synapse formation and maintenance, it is desirable to identify the factors involved and establish methods for inducing them. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sudhof TC, Towards an Understanding of Synapse Formation. Neuron. 2018 Oct 24;100(2):276-293 [Non-Patent Document 2] Uemura et al., Trans-synaptic interaction of GluRdelta2 and Neurexin through Cbln1 mediates synapse formation in the cerebellum. Cell. 2010 Jun 11;141(6):1068-79. [Non-Patent Document 3] Siddiqui et al., An LRRTM4-HSPG complex mediates excitatory synapse development on dentate gyrus granule cells. Neuron. 2013 Aug 21;79(4):680-95. [Non-Patent Document 4] Yumoto et al., Lrp4 is a retrograde signal for presynaptic differentiation at neuromuscular synapses. Nature. 2012 Sep 20;489(7416):438-42. [Non-Patent Document 5] Linhoff et al., An unbiased expression screen for synaptogenic proteins identifies the LRRTM protein family as synaptic organizers. Neuron. 2009 Mar 12;61(5):734-49. [Non-Patent Document 6] J. Ko et al., LRRTM2 Function as a Neurexin Ligand in Promoting Excitatory Synapse Formation, Neuron 64, 791-798 (2009) [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention aims to identify factors that induce presynaptic terminal formation in human nerve cells and to provide a method for inducing presynaptic terminal formation in human nerve cells using these factors. [Means for solving the problem]

[0009] As a result of diligent research, the inventors discovered that LRRTM2 (Leucine Rich Repeat Transmembrane Protein 2), a molecule in the LRRTM family, induces presynaptic formation in human nerve cells. Furthermore, by co-culturing nerve cells with microbeads immobilized with LRRTM2 via a specific linker, they succeeded in inducing presynaptic formation in nerve cells, thus completing the present invention. The present invention includes the following embodiments.

[0010] (1) A method for inducing presynaptic formation in mammalian nerve cells (preferably human nerve cells), comprising co-culturing the nerve cells with microbeads on which at least one LRRTM molecule selected from the group consisting of LRRTM (Leucine-rich repeat transmembrane neuronal protein) family molecules or a fusion protein containing such molecule is immobilized on the surface (wherein the LRRTM molecule or the fusion protein containing such molecule is immobilized on the surface of the microbeads via a linker). (2) The method according to (1) above, wherein the fusion protein containing the LRRTM molecule is a fusion protein containing the LRRTM molecule and the Fc region of IgG (preferably human Ig)G, the linker is an anti-IgGFc antibody (preferably anti-human IgGFc antibody) immobilized on the surface of the microbead, and the fusion protein containing the LRRTM molecule is immobilized on the surface of the microbead via binding between the Fc region of IgG (preferably human IgG) and the anti-IgGFc antibody (preferably anti-human IgGFc antibody). (3) The method according to (1) above, wherein the linker is a polymer (for example, a protein, a modified polyethylene glycol, a modified sugar chain, and a modified nucleic acid, etc.) and the length of the linker is 10 nm or more. (4) The method according to (1) above, wherein the distance between the LRRTM molecule fixed to the surface of the microbead or the LRRTM molecule which is part of the fusion protein and the surface of the microbead is 10 nm or more. (5) The method according to any one of (1) to (4) above, wherein the nerve cells are human peripheral nerve cells. (6) The method according to any one of (1) to (4) above, wherein the nerve cells are human central nervous system cells. (7) The method according to (5) above, wherein the nerve cells are human motor nerve cells. (8) The method according to (6) above, wherein the nerve cells are human glutamatergic nerve cells. (9) The method according to any one of (1) to (8) above, wherein the LRRTM molecule is an LRRTM1 molecule, an LRRTM2 molecule, an LRRTM3 molecule, or an LRRTM4 molecule. (10) The method according to (9) above, wherein the LRRTM molecule is LRRTM2. (11) The method according to any one of (1) to (10) above, wherein the nerve cells are nerve cells differentiated from human-derived pluripotent stem cells. (12) The method according to (11) above, wherein the nerve cells are nerve cells differentiated from human iPS cells or human ES cells. (13) The method according to any one of (1) to (12) above, wherein the fusion protein is a fusion protein obtained by expressing in a host a plasmid containing DNA that encodes the amino acid sequence of an LRRTM molecule and DNA that encodes the amino acid sequence of the Fc region of human IgG. (14) The method according to (13) above, wherein the LRRTM molecule is LRRTM2. (15) The method according to any one of (1) to (14) above, further comprising the step of immunostaining the presynaptic portion of cultured cells with an anti-synapsin antibody to confirm the induction of presynaptic formation. (16) Furthermore, after culturing, detect one of the following: (a) To detect neurotransmitters released from synapses, (b) detecting the expression of proteins related to the release of neurotransmitters from synapses, or (c) Add a labeling agent (e.g., a dye molecule) that labels synaptic vesicles to the culture medium, and visualize and detect synaptic vesicles that are reuptaken after the release of neurotransmitters from the synapse. The method according to any one of (1) to (14) above, further comprising the step of confirming that the synapse, including the induced presynaptic portion, is functional.

[0011] (17) Microbeads for use in culturing mammalian nerve cells (preferably human nerve cells) to induce synapse formation, having at least one LRRTM molecule selected from the group consisting of LRRTM (Leucine-rich repeat transmembrane neuronal protein 2) family molecules or a fusion protein containing such molecule immobilized on the surface via a linker. (18) The microbeads according to (17) above, wherein the fusion protein is a fusion protein comprising an LRRTM molecule and the Fc region of IgG (preferably human IgG), the linker is an anti-IgGFc antibody (preferably anti-human IgGFc antibody) immobilized on the surface of the microbeads, and the fusion protein comprising the LRRTM molecule is immobilized on the surface of the microbeads via binding between the Fc region of IgG (preferably human IgG) and the anti-IgGFc antibody (anti-human IgGFc antibody). (19) The microbeads according to (17) above, wherein the linker is a polymer (for example, a protein, a modified polyethylene glycol, a modified sugar chain, and a modified nucleic acid, etc.) and the length of the linker is 10 nm or more. (20) The microbeads described in (17) above, wherein the distance between the LRRTM molecule fixed to the surface of the microbead or the LRRTM molecule which is part of a fusion protein and the surface of the microbead is 10 nm or more. (21) The microbeads according to any one of (17) to (20) above, wherein the nerve cells are human peripheral nerve cells. (22) Microbeads according to any one of (17) to (20) above, wherein the nerve cells are human central nervous system cells. (23) The microbeads according to (21) above, wherein the nerve cells are human motor nerve cells. (24) The microbeads according to (22) above, wherein the nerve cells are human glutamatergic nerve cells. (25) The microbeads according to any one of (17) to (24) above, wherein the nerve cells are nerve cells differentiated and induced from human-derived pluripotent stem cells. (26) The microbeads according to (25) above, wherein the human nerve cells are nerve cells differentiated and induced from human iPS cells or human ES cells.

[0012] (27) A method for screening a drug for a nerve disease, comprising the following steps: (i) Co-culturing nerve cells differentiated and induced from iPS cells derived from a patient suffering from a nerve disease with microbeads having at least one LRRTM molecule selected from the group consisting of LRRTM (Leucine-rich repeat transmembrane neuronal protein) family molecules or a fusion protein containing the molecule immobilized on the surface thereof to induce the formation of presynaptic terminals (wherein the LRRTM molecule or the fusion protein containing the molecule is immobilized on the surface of the microbeads via a linker), (ii) Adding a target substance to the culture medium in step (i) or after step (i) and culturing, and (iii) Then, detecting the effect of the target substance on the formation of presynaptic terminals in the nerve cells. A screening method comprising the above steps. (28) The detection is (a) Observing the morphological state of the formed presynaptic terminals, (b) Detecting neurotransmitters released from the presynaptic terminals (c) Detecting the expression of proteins related to the release of neurotransmitters from synapses, and (d) Adding a labeling agent (for example, a dye molecule) for labeling synaptic vesicles to the culture medium and visualizing and detecting synaptic vesicles that are re-uptaken after the release of neurotransmitters from synapses. The method according to (27) above, which is performed by at least one selected from the above. (29) The method according to (27) or (28) above, wherein the fusion protein containing the LRRTM molecule is a fusion protein containing the LRRTM molecule and the Fc region of human IgG, the linker is an anti-human IgGFc antibody immobilized on the surface of a microbead, and the fusion protein containing the LRRTM molecule is immobilized on the surface of a microbead via binding between the Fc region of human IgG and the anti-human IgGFc antibody. (30) The method according to (27) or (28) above, wherein the linker is a polymer (for example, a protein, a modified polyethylene glycol, a modified sugar chain, and a modified nucleic acid, etc.) and the length of the linker is 10 nm or more. (31) The method according to any one of the above (27) to (30), wherein the LRRTM molecule is LRRTM1, LRRTM2, LRRTM3, or LRRTM4. (32) The method according to (31) above, wherein the LRRTM molecule is LRRTM2. (33) The method according to any one of the above (27) to (32), wherein the neurological disorder is a motor neuron disorder, a neuromuscular disorder, or a mental disorder. (34) The method according to any one of (27) to (32) above, wherein the neurological disorder is a neurological disorder selected from the group consisting of amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), myasthenia gravis, Lambert-Eaton syndrome, Alzheimer's disease, dementia such as frontotemporal dementia, epilepsy, Parkinson's disease, schizophrenia, autism, autism spectrum disorder, and other mental disorders. (35) A screening method which is performed by high-throughput screening (HTS) using any one of the methods described in (27) to (34) above.

[0013] (36) A method for screening substances that promote or inhibit the release of neurotransmitters, comprising the following steps: (a) A step of co-culturing mammalian nerve cells (preferably human nerve cells) with microbeads on which at least one LRRTM molecule selected from the group consisting of LRRTM (Leucine-rich repeat transmembrane neuronal protein) family molecules or a fusion protein containing that molecule is immobilized on the surface to induce presynaptic formation (wherein the LRRTM molecule or the fusion protein containing that molecule is immobilized on the surface of the microbeads via a linker), (b) Next, the process of culturing nerve cells with the target substance added to the culture medium, (c) The following detection steps: (c-1) A step to detect neurotransmitters released into the culture medium, (c-2) A step of detecting the expression of proteins related to the release of neurotransmitters from synapses, and (c-3) A step of adding a labeling agent (e.g., a dye molecule) that labels synaptic vesicles to the culture medium, and visualizing and detecting synaptic vesicles that are reuptaken after the release of neurotransmitters from the synapse. One of the processes selected from the group consisting of, A screening method that includes [this]. (37) The method according to (36), wherein in step (b), a substance that stimulates synapses and induces the release of neurotransmitters is further added to the culture medium. (38) The method according to (36) or (37) above, wherein the fusion protein containing the LRRTM molecule is a fusion protein containing the LRRTM molecule and the Fc region of IgG (preferably human IgG), the linker is an anti-IgGFc antibody (preferably anti-human IgGFc antibody) immobilized on the surface of a microbead, and the fusion protein containing the LRRTM molecule is immobilized on the surface of a microbead via binding between the Fc region of IgG (preferably human IgG) and the anti-IgGFc antibody (preferably anti-human IgGFc antibody). (39) The method according to (36) or (37), wherein the linker is a polymer (for example, a protein, a modified polyethylene glycol, a modified sugar chain, and a modified nucleic acid, etc.) and the length of the linker is 10 nm or more. (40) The method according to any one of (36) to (39) above, wherein the nerve cells are human peripheral nerve cells. (41) The method according to any one of the above (36) to (39), wherein the nerve cells are human central nervous system cells. (42) The method according to (40) above, wherein the nerve cells are human motor nerve cells. (43) The method according to (41) above, wherein the nerve cells are human glutamatergic nerve cells. (44) The method according to any one of the above (36) to (43), wherein the LRRTM molecule is LRRTM1, LRRTM2, LRRTM3, or LRRTM4. (45) The method according to (44) above, wherein the LRRTM molecule is LRRTM2. (46) The method according to any one of (36) to (45) above, wherein the nerve cells are nerve cells differentiated from human-derived pluripotent stem cells. (47) The method according to (45) above, wherein the human nerve cells are nerve cells differentiated from human iPS cells or human ES cells. (48) The method according to any one of (36) to (47) above, wherein the fusion protein is a fusion protein obtained by expressing in a host a plasmid containing DNA encoding the amino acid sequence of an LRRTM molecule and DNA encoding the amino acid sequence of the Fc region of human IgG. (49) The method according to (47) above, wherein the LRRTM molecule is LRRTM2.

[0014] (50) A method for diagnosing whether a subject (preferably a human) is suffering from a disease, (I) A step of co-culturing nerve cells differentiated from iPS cells derived from the target with microbeads on which at least one LRRTM molecule selected from the group consisting of LRRTM (Leucine-rich repeat transmembrane neuronal protein) family molecules or a fusion protein containing that molecule is immobilized on the surface to induce presynaptic formation (wherein the LRRTM molecule or the fusion protein containing that molecule is immobilized on the surface of the microbeads via a linker), (II) One detection step selected from the group consisting of (a) to (c) below: (a) A step of detecting neurotransmitters contained in a synapse or neurotransmitters released from a synapse into a culture medium, (b) A step of detecting the expression of proteins related to the release of neurotransmitters from synapses, and (c) Adding a labeling agent (e.g., a dye molecule) to the culture medium to visualize and detect synaptic vesicles that are reuptaken after the release of neurotransmitters from the synapse, and (III) A step of diagnosing that a subject is suffering from a specific disease based on the relationship between the specific disease and the results detected by the detection step, Diagnostic methods including those mentioned above. (51) The method according to (50), wherein step (II) is a step of (II-1) adding a substance that stimulates synapses and induces the release of neurotransmitters to a culture medium, and (II-2) detecting neurotransmitters released from synapses into the culture medium. (52) The method according to (50) or (51) above, wherein the fusion protein containing the LRRTM molecule is a fusion protein containing the LRRTM molecule and the Fc region of human IgG, the linker is an anti-IgGFc antibody (preferably an anti-human IgGFc antibody) immobilized on the surface of a microbead, and the fusion protein containing the LRRTM molecule is immobilized on the surface of a microbead via binding between the Fc region of human IgG and the anti-IgGFc antibody (preferably an anti-human IgGFc antibody). (53) The method according to (50) or (51), wherein the linker is a polymer (for example, a protein, a modified polyethylene glycol, a modified sugar chain, and a modified nucleic acid, etc.) and the length of the linker is 10 nm or more. (54) The method according to any one of (50) to (53) above, wherein the nerve cells are human peripheral nerve cells. (55) The method according to any one of the above (50) to (53), wherein the nerve cells are human central nervous system cells. (56) The method according to (54) above, wherein the nerve cells are human motor nerve cells. (57) The method according to (55) above, wherein the nerve cells are human glutamatergic nerve cells. (58) The method according to any one of the above (50) to (57), wherein the LRRTM molecule is LRRTM1, LRRTM2, LRRTM3, or LRRTM4. (59) The method according to (58) above, wherein the LRRTM molecule is LRRTM2. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a schematic representation of microbeads on which LRRTM2 molecules are immobilized, which is one embodiment of the present invention. [Figure 2] This study confirmed the formation of presynaptic terminals when LRRTM2-Fc-anti-human IgGFc antibody microbeads (LRRTM2-Fc Linker(+)) or the control Fc-anti-human IgGFc antibody microbeads (Control-Fc Linker(+)) were co-cultured with glutamatergic neurons. [Figure 3] This study confirmed the formation of presynaptic terminals when LRRTM2-Fc-anti-human IgGFc antibody microbeads (LRRTM2-Fc Linker(+)) or LRRTM2-Fc-microbeads (LRRTM2-Fc Linker(-)) were co-cultured with glutamatergic neurons. [Figure 4]This result shows the percentage of beads that exhibited positive synapsin induction activity when synapse formation was induced using LRRTM2-Fc-anti-human IgGFc antibody microbeads, which were created by progressively reducing the amount of LRRTM2-Fc immobilized on the beads. 100% represents the result for beads prepared using an LRRTM2 solution at a concentration of 50 μg / mL, and 10% represents the result for beads prepared using an LRRTM2 solution at 1 / 10th of that concentration. 0% represents the control. [Figure 5] This study confirmed the formation of presynaptic terminals when LRRTM2-Fc-anti-human IgGFc antibody microbeads (LRRTM2-Fc Linker(+)) or the control Fc-anti-human IgGFc antibody microbeads (Control-Fc Linker(+)) were co-cultured with motor neurons. [Figure 6] This study involved co-culturing LRRTM2-Fc-anti-human IgGFc antibody microbeads (LRRTM2-Fc Linker(+)) with motor neurons and confirming presynaptic formation using the marker VAChT (vesicular acetylcholine transporter). [Figure 7] This study confirmed the formation of presynaptic terminals when RRTM2-Fc-anti-human IgGFc antibody microbeads (LRRTM2-Fc Linker(+)) or LRRTM2-Fc-microbeads (LRRTM2-Fc Linker(-)) were co-cultured with motor neurons. [Figure 8] This is the result of forming presynaptic terminals from motor neurons induced from iPS cells derived from healthy individuals or ALS patients using the method of the present invention, and confirming the expression of synapsin and acetylcholine transporter (VAChT). [Figure 9]This report presents the results of examining the effects of 1814 compounds listed in the FDA-approved drug compound library on synapse formation using the screening method of the present invention. Motor neurons were co-cultured with LRRTM2-Fc-anti-human IgGFc antibody microbeads (Positive) or the control Fc-anti-human IgGFc antibody microbeads (Negative). After forming presynaptic terminals in the presence of various compounds, immunostaining was performed with a presynaptic marker (synapsin 1), and the difference in fluorescence intensity (SSMD, strictly standardized mean difference) compared to the experimental control is shown. [Modes for carrying out the invention]

[0016] The present invention will be described below, with illustrative embodiments as examples, along with preferred methods and materials that may be used in carrying out the invention. Unless otherwise specified herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention pertains. Any materials and methods equivalent to or similar to those described herein may be used in carrying out the invention. Furthermore, all publications and patents referenced herein in connection with the invention constitute part of this specification, for example, as indicating methods, materials, and other things that may be used in the invention. In this specification, "approximately" means that a tolerance of ±10% is allowed.

[0017] (1) Nerve cells The nerve cells that can be used in the method of the present invention are nerve cells of mammals. Examples of mammals include primates (e.g., humans), cats, dogs, cows, sheep, goats, horses, rabbits, rats, mice, and koalas, but humans are preferred. The cells that can be used in the method of the present invention are preferably human nerve cells. The present invention will be described below using human nerve cells as an example, but the present invention is not limited to human nerve cells. Examples of human nerve cells include human peripheral nerve cells and human central nervous system cells, and any of these cells can be used in the present invention. Human nerve cells can be used without restriction, regardless of their origin. For example, but not limited to these, examples include primary cultures of cells isolated from humans, cells isolated from humans and established as cell lines, and human nerve cells differentiated from human-derived pluripotent stem cells. Preferably, human-derived pluripotent stem cells are used. The term "pluripotent stem cells" used in the present invention refers to cells that have the ability to self-renew, can be cultured in vitro, and have the multipotency to differentiate into cells that constitute an individual. Specifically, examples include embryonic stem cells (ES cells), pluripotent stem cells (GS cells) derived from primordial germ cells of a fetus, and induced pluripotent stem cells (iPS cells) derived from somatic cells. However, in the present invention, human-derived iPS cells or ES cells are preferred, and human-derived iPS cells are particularly preferred.

[0018] ES cells can generally be established as a cell line by culturing blastocyst-stage fertilized eggs with feeder cells, separating the cells derived from the proliferated inner cell mass, and repeatedly subplanting them. Thus, ES cells are often obtained from fertilized eggs, but they can also be obtained from other sources, such as adipose tissue, placenta, and testicular cells, and any type of ES cell is within the scope of this invention. Methods for creating ES cells from sources other than fertilized eggs have been reported, and these reports can be used by referring to them as appropriate. For example, but not limited to, the disclosure in WO2003 / 046141 can be cited.

[0019] iPS cells are artificial stem cells derived from somatic cells that can be produced by introducing specific reprogramming factors into somatic cells in the form of nucleic acids or proteins, and exhibit characteristics almost equivalent to those of ES cells (e.g., pluripotency and proliferative ability based on self-renewal). Reprogramming factors may consist of genes specifically expressed in ES cells, their gene products or their non-coding RNAs, genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products or their non-coding RNAs, or low molecular weight compounds. Examples of genes included in reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-MYC, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, etc. These reprogramming factors may be used individually or in combination. When using the c-MYC gene as a reprogramming factor by introducing it into somatic cells, it is preferable to use an introduction method that minimizes the likelihood of the introduced c-MYC gene being incorporated into the target cell's chromosome after iPS cell creation. Examples include, but are not limited to, introduction using a Sendai virus vector or an episomal vector. Many reports have been made on methods for producing iPS cells, and these reports can be referred to and modified as appropriate. The iPS cells that can be used in this invention are preferably human-derived iPS cells, for example, human fibroblast-derived iPS cells.

[0020] In this invention, peripheral nerve cells or central nervous cells differentiated from human iPS cells are preferably used. Many methods have been reported for differentiating human iPS cells into peripheral nerve cells, and these reports can be referenced and modified as appropriate. For example, a method for differentiating human iPS cells into peripheral nerve cells, such as motor nerve cells, can be found in Chambers, Stuart M., et al. "Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling." Nature biotechnology 27.3 (2009): 275. Many methods have also been reported for differentiating human iPS cells into central nervous cells, and these reports can be referenced and modified as appropriate. For methods of differentiating human iPS cells into central nervous system cells, such as glutamatergic neurons, see, for example, Shi, Yichen, et al. "Human cerebral cortex development from pluripotent stem cells to functional excitatory synapses." Nature neuroscience 15.3 (2012): 477. Alternatively, various types of neurons differentiated from human iPS cells are commercially available and can be purchased from companies such as ReproCELL, Inc.

[0021] The method of the present invention allows for the study of presynaptic formation in peripheral nerve cells, making it possible to confirm whether a patient's disease is related to synapse formation by using peripheral nerve cells differentiated from iPS cells derived from patients with peripheral nerve disease. Furthermore, by evaluating the functionality of the formed synapses, it becomes possible to confirm whether the patient's disease is related to synaptic function (for example, but not limited to, the ability to release neurotransmitters). Examples of peripheral nerve diseases, but not limited to, include motor neuron diseases and neuromuscular diseases such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), myasthenia gravis, and Lambert-Eaton syndrome.

[0022] The human peripheral nerve cells used in the method of the present invention are not limited to these, but include, for example, human motor nerve cells, human sensory nerve cells, human sympathetic nerve cells, and human parasympathetic nerve cells, and are preferably human motor nerve cells.

[0023] The method of the present invention allows for the study of presynaptic formation in central nervous system cells (CNS), making it possible to determine whether a patient's disease is related to synapse formation by using CNS differentiated from iPS cells derived from patients with CNS diseases. Furthermore, by evaluating the functionality of the formed synapses, it becomes possible to determine whether the patient's disease is related to synaptic function (for example, but not limited to, the ability to release neurotransmitters). Examples of CNS diseases, but not limited to, include dementias such as Alzheimer's disease and frontotemporal dementia, epilepsy, Parkinson's disease, schizophrenia, autism, autism spectrum disorder, and various mental illnesses.

[0024] The human central nervous system cells used in the method of the present invention are not limited to these, but include, for example, human glutamatergic neurons, cholinergic neurons, adrenergic neurons, dopaminergic neurons, serotonergic neurons, and noradrenergic neurons, and are preferably human glutamatergic neurons.

[0025] According to the method of the present invention, presynaptic terminals can be formed using nerve cells, and functional synapses can be formed. Synapse formation can be confirmed, for example, by observing the morphology of the formed synapse, or by detecting proteins expressed at the synapse, i.e., presynaptic markers (e.g., synapsin 1). Since neurotransmitters are released from functional synapses, it is possible to detect neurotransmitters using the method of the present invention, and furthermore, it can be used to detect substances that promote or inhibit neurotransmitter release. The ability to release neurotransmitters from a synapse may be determined by directly detecting the substances actually released from the synapse, or by detecting various proteins related to neurotransmitter release. Substances released from synapses, i.e., neurotransmitters, include, but are not limited to, amino acids (e.g., glutamic acid, γ-aminobutyric acid, aspartic acid, glycine), peptides (e.g., vasopressin, gastrin, somatostatin, neurotensin, neuropeptides, opioids, secretin, tachykinin), monoamines (e.g., dopamine, norepinephrine, octopamine, tyramine, phenylamine, phenylatanolamine, serotonin, histamine), and acetylcholine. Proteins involved in the release of neurotransmitters from synapses can be detected by detecting the expression of synaptic vesicle-related molecules such as vesicular acetylcholine transporter (VAChT), vesicular glutamate transporter (VGlut), vesicular monoamine transporter (VMAT), vesicular GABA transporter (VGAT), synapsin, synaptotagmin, synaptophysin, SNAP25, and active zone markers such as Bassoon and Piccolo. In detecting neurotransmitter release, synapses may be stimulated as needed, and neurotransmitters are released from the synapses in response to the stimulation. For example, although not limited to this, after forming the presynaptic terminal in nerve cells using the method of the present invention, the neurotransmitters released into the culture supernatant can be detected after stimulation.The stimulus is not particularly limited as long as it releases neurotransmitters, but examples include stimulation by various drugs, compounds, electrodes, etc. The detection of neurotransmitters is not particularly limited, and any method that can detect the target substance can be used as appropriate. For example, the target neurotransmitter can be detected using various methods such as ELISA, various mass spectrometry methods (e.g., LC-MS, tandem LC-MS), and enzyme reaction methods, but ELISA is preferred. This makes it possible to evaluate the functionality of synapses induced by the method of the present invention, and to screen for compounds that stimulate synapses and act on the release of neurotransmitters.

[0026] Functional synapse formation can also be confirmed by visualizing and detecting synaptic vesicles, which are reabsorbed after the release of neurotransmitters from the synapse, using a labeling substance that has been pre-added to the extracellular space. While not limited to these, labeling substances for synaptic vesicles include, for example, dye molecules and FM dyes (FM TM Examples of such labels include 4-64 Dye (N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino)Phenyl)Hexatrienyl)Pyridinium Dibromide) and Thermo Fisher. By adding these labeling substances to the culture medium and culturing cells, the formation of functional synapses can be confirmed.

[0027] Using the method of the present invention, it is also possible to select substances that promote or inhibit the release of neurotransmitters under conditions that mimic the conditions in vivo. Conditions that mimic the conditions in vivo include, for example, a method of culturing cells in a state in which the axon and synapse-forming portion of nerve cells can be identified as separate structures. In carrying out the method of the present invention, various cell culture devices can be used, modified or changed as appropriate, but preferably the device described in WO2017 / 187696 is used, and by using this device, functional synapse formation can be successfully achieved. Using the method of the present invention, functional synapses are formed in nerve cells, and then, by stimulating the nerve cells in the same manner as in vivo, neurotransmitters are released into the culture supernatant. The neurotransmitters released into the culture supernatant can be detected and quantified by, for example, mass spectrometry, ELISA, or enzyme reaction methods. In such measurement systems, by adding various substances to the culture medium, substances that promote or inhibit the release of neurotransmitters can be selected. Therefore, the present invention can also be used as a screening method for compounds that affect the release of neurotransmitters under conditions that mimic those found in living organisms.

[0028] There are no particular restrictions on the neurotransmitters to be detected, but examples include acetylcholine, amino acids, monoamines, and neuropeptides (polypeptides) as mentioned above. Acetylcholine is released in response to stimuli from synapses at the terminals of motor neurons of cholinergic nerves. Therefore, although not limited to this, the method of the present invention can be used to form functional synapses in nerve cells, and then acetylcholine is released into the culture supernatant by stimulating the nerve cells (e.g., glutamate, high concentration potassium, 4-aminopyrrolidone, etc.). The acetylcholine released into the culture supernatant can be detected and quantified by, for example, mass spectrometry, ELISA, or an enzymatic reaction method using cholinesterase. ELISA is preferred. In addition, by adding various substances to the culture medium, it is also possible to select substances that promote or inhibit the release of acetylcholine. For example, although not limited to this, such evaluation methods include the evaluation of acetylcholine neurotransmitter enhancers and the evaluation of potassium ion channel and calcium ion channel inhibitors or activators.

[0029] According to the method of the invention, functional synapses can be formed using nerve cells, and neurotransmitters are released from the synapses. Therefore, abnormalities in neurotransmitter release can be detected using the method of the present invention. Thus, by using the method of the present invention, it becomes possible to diagnose diseases based on the relationship between the release of specific neurotransmitters and specific diseases. For example, after differentiating iPS cells derived from a target patient into nerve cells, synapses can be formed using these nerve cells according to the present invention. Then, by detecting specific neurotransmitters released from the synapses (e.g., the above-mentioned neurotransmitters), or by detecting the expression of proteins associated with the release of specific neurotransmitters (e.g., the above-mentioned related proteins) at the synapses, or by visualizing and detecting synaptic vesicles, which are reabsorbed after the release of neurotransmitters from the synapses, using a labeling substance (e.g., a dye molecule) that has been pre-added extracellularly, it becomes possible to determine whether the subject is suffering from a specific disease related to abnormal synapse formation or the release of neurotransmitters. Thus, the present invention is also a method for diagnosing diseases based on any of the above detection methods.

[0030] (2) LRRTM (Leucine-rich repeat transmembrane neuronal protein) molecule The LRRTM molecule used in the present invention is a molecule called the LRRTM family. The LRRTM family is one of the synaptic organizer molecule families on the postsynaptic terminal side, and four types have been reported in humans: LRRTM1, LRRTM2, LRRTM3, and LRRTM4. In the present invention, any of these four types of LRRTM molecules can be used, but LRRTM2 is preferred. The LRRTM molecule, which is one of the synaptic adhesion molecules, is a single-pass transmembrane protein and contains an extracellular domain and an internal domain. The LRRTM molecule that can be used in the present invention is a molecule that contains at least an extracellular domain. Therefore, for example, in the present invention, the human LRRTM1 molecule means a human LRRTM1 molecule that has at least the amino acid sequence from the 1st to the 392nd amino acid of SEQ ID NO: 1, which is its extracellular domain.

[0031] Human LRRTM1 is a membrane protein consisting of 488 amino acids, as shown in SEQ ID NO: 1. After translation, it is expressed as a protein consisting of 522 amino acids with a signal peptide consisting of 34 amino acids. The extracellular domain of LRRTM1 has been reported to be from the 1st to the 392nd amino acid sequence shown in SEQ ID NO: 1, and this region is considered important for inducing presynaptic terminal formation. The LRRTM1 molecule used in this invention is not limited to the protein having the 488 amino acid sequence shown in SEQ ID NO: 1, but means a protein containing the 1st to the 392nd amino acids of the amino acid sequence shown in SEQ ID NO: 1 that has presynaptic terminal formation inducing activity, and as long as it has the activity to induce presynaptic terminal formation in nerve cells, it may be the case that a portion of the sequence (for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of the entire sequence) is substituted, deleted, or added.

[0032] Human LRRTM2 is a membrane protein consisting of 483 amino acids, as shown in SEQ ID NO: 2. After translation, it is expressed as a 516-amino acid protein containing a signal peptide of 33 amino acids. The extracellular domain of LRRTM2 has been reported to be from the 1st to the 389th amino acid of the amino acid sequence shown in SEQ ID NO: 2, and this region is considered important for inducing presynaptic terminal formation. The LRRTM2 molecule used in this invention is not limited to the protein with the 483-amino acid sequence shown in SEQ ID NO: 2, but refers to a protein containing the 1st to the 389th amino acids of the amino acid sequence shown in SEQ ID NO: 2 that has presynaptic terminal formation inducing activity. As long as it has the activity to induce presynaptic terminal formation in nerve cells, it may be the case that a portion of these sequences (for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of the entire sequence) is substituted, deleted, or added.

[0033] Human LRRTM3 is a membrane protein consisting of 551 amino acids, as shown in Sequence ID No. 3. After translation, it is expressed as a protein consisting of 581 amino acids with a signal peptide consisting of 30 amino acids. The extracellular domain of LRRTM3 has been reported to be from the 1st to the 389th amino acid sequence shown in Sequence ID No. 3, and this region is considered important for inducing presynaptic terminal formation. The LRRTM3 molecule used in this invention is not limited to the protein having the 551 amino acid sequence shown in Sequence ID No. 3, but means a protein containing the 1st to the 389th amino acids of the amino acid sequence shown in Sequence ID No. 3 that has presynaptic terminal formation inducing activity, and as long as it has the activity to induce presynaptic terminal formation in nerve cells, it may be the case that a portion of the sequence (for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of the entire sequence) is substituted, deleted, or added.

[0034] Human LRRTM4 is a membrane protein consisting of 560 amino acids, as shown in SEQ ID NO: 4. After translation, it is expressed as a protein consisting of 590 amino acids with a signal peptide consisting of 30 amino acids. The extracellular domain of LRRTM4 has been reported to be from the 1st to the 394th amino acid of the amino acid sequence shown in SEQ ID NO: 4, and this region is considered important for inducing presynaptic terminal formation. The LRRTM4 molecule used in this invention is not limited to the protein with the 560 amino acid sequence shown in SEQ ID NO: 4, but means a protein containing the amino acids from the 1st to the 394th amino acid of the amino acid sequence shown in SEQ ID NO: 4 that has presynaptic terminal formation inducing activity, and as long as it has the activity to induce presynaptic terminal formation in nerve cells, it may be the case that a portion of the sequence (for example, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of the entire sequence) is substituted, deleted, or added.

[0035] (3) Fusion protein The fusion protein containing the LRRTM molecule used in the present invention is a fusion protein containing any of the LRRTM molecules described above. The protein portion of the fusion protein other than the LRRTM molecule can be arbitrarily selected as long as it does not inhibit the formation of the presynaptic terminal, which is the objective of the present invention. By using the fusion protein, binding to the linker and providing distance between the LRRTM molecule and the microbead surface can be achieved, although this is not limited to the fusion protein. Preferably, the fusion protein is a fusion protein containing the LRRTM molecule and the Fc region of human IgG. Therefore, a preferred fusion protein used in the present invention is a fusion protein containing at least one of these four types of LRRTM molecules and the Fc region of human IgG, and particularly preferably, a fusion protein containing the human LRRTM2 (Leucine-rich repeat transmembrane neuronal protein 2) molecule and the Fc region of human IgG.

[0036] The following explanation will describe a fusion protein containing the Fc region of human IgG and an LRRTM molecule, using the LRRTM2 molecule as an example. However, it will be readily apparent to those skilled in the art that the same principles apply to other LRRTM molecules. Fusion proteins of the human IgG Fc region and the LRRTM2 molecule can be produced by appropriately referring to known methods. However, they can also be produced by preparing a vector containing DNA encoding the 483-amino acid sequence of human LRRTM2 and DNA encoding the amino acid sequence of the human IgG Fc region, and then transforming and expressing this vector in any host used for recombinant protein expression, such as E. coli, yeast, insect cells, or mammalian cultured cells. If glycosylation is desired, it can be produced by appropriately selecting a host capable of producing glycosylated recombinant proteins according to conventional methods. The ligation of the DNA encoding human LRRTM2 and the DNA encoding the human IgG Fc region can be performed using conventional methods, and any peptide sequence can be inserted between them during ligation. Furthermore, the order of the two DNA sequences does not matter as long as the resulting fusion protein exhibits inductive activity when immobilized on microbeads. The expressed fusion protein may be in the order of human LRRTM2 (-any peptide sequence)-human IgG Fc region, or human IgG Fc region (-any peptide sequence)-human LRRTM2, viewed from the N-terminus. The resulting fusion protein can be purified using known methods to obtain a fusion protein containing the LRRTM molecule used in this invention. Fusion proteins of the Fc region of human LRRTM2 and human IgG are also commercially available and can be used. For example, they are sold by R&D Systems.

[0037] The fusion protein may include the LRRTM2 molecule and the Fc region of human IgG, as well as any peptide linking the LRRTM2 molecule and the Fc region of human IgG (e.g., but not limited to, peptides consisting of 1-100, 3-80, 5-80, 5-50, 5-30, and 5-20 amino acids), any peptide constituting the N-terminus (e.g., but not limited to, peptides consisting of 1-50, 1-30, 2-20, and 2-10 amino acids), and any peptide constituting the C-terminus (e.g., but not limited to, peptides consisting of 1-50, 1-30, 2-20, and 2-10 amino acids).

[0038] (4) Linker The linker that can be used in the present invention is any substance that can link the LRRTM molecule or a fusion protein containing it with the microbeads. Examples include polymers such as proteins, modified polyethylene glycol (PEG), modified glycans, and modified nucleic acids, but preferably proteins or modified PEG. Modified PEG refers to PEG with any substituent attached at any position. Many reports have been made on the modification of PEG, and these can be referenced as appropriate. Modified glycans refer to glycans with any substituent attached at any position. Many methods for modifying glycans have been reported, and these can be referenced as appropriate. There are no particular restrictions on the type of glycan; for example, it is a glycan composed of one or more types of sugars selected from pentose or hexose sugars, and the glycan may be linear or branched. The nucleic acid can be either DNA or RNA, and the ribonucleotides / ribonucleosides that make up DNA / RNA may be modified. By using a linker, however limited, LRRTM molecules can be immobilized onto microbeads, and a distance can be provided between the LRRTM molecules and the microbead surface, thereby providing mobility of the LRRTM molecules in three-dimensional space. Therefore, the linker preferably has a reactive group and a certain length. The length of the linker is preferably 10 nm or more, more preferably 15 nm or more, and even more preferably 20 nm or more. Furthermore, since it is desirable for a certain number of LRRTM molecules to be immobilized on the surface of the microbeads in order to induce presynaptic formation, a linker that can bind to multiple LRRTM molecules or a fusion protein containing multiple LRRTM molecules is preferably used.

[0039] (5) Linking with the linker The linking of the LRRTM molecule to the linker can be carried out by appropriately referring to known methods. For example, it can be done by chemically linking the end of the LRRTM molecule or the side chains of the amino acids that make up the molecule to the linker. For example, it can be chemically linked by reacting an amine or carboxyl group with a reactive group present in the linker. If the linker is a protein, known methods used for linking proteins together can be used. If the linker is PEG, which may be modified, known methods for PEG modification of proteins can be used. If the linker is a sugar chain, it can be linked by reacting an aldehyde group obtained by oxidizing the sugar chain with a group present in the LRRTM molecule, such as an amine.

[0040] The binding of a fusion protein containing the LRRTM molecule to a linker can be carried out by appropriately referring to known methods depending on the type of fusion protein and the type of linker. The methods described above are examples of methods for chemically binding the fusion protein and the linker. Furthermore, for example, if the fusion protein contains the LRRTM molecule and the Fc region of human IgG, the Fc region of IgG can be chemically bound to the linker, or a human anti-IgGFc antibody can be used as the linker, and the fusion protein and the linker (anti-IgGFc antibody) can be bound by an antigen-antibody reaction.

[0041] (6) Microbeads The microbeads that can be used in this invention are not particularly limited as long as they do not particularly affect the survival of nerve cells when cultured together with nerve cells. Examples include microbeads made of polyethylene, silica, or magnetic beads. The size of the microbeads is not particularly limited, but for example, the average diameter can be 1 to 50 μm, preferably 5 to 30 μm, more preferably 5 to 20 μm, even more preferably 5 to 15 μm, and most preferably about 10 μm.

[0042] The microbeads used in this invention have an LRRTM molecule or a fusion protein containing that molecule immobilized on their surface via a linker. The linker can be attached to the microbead surface using conventional methods, depending on the type of linker. For example, if microbeads with protein A immobilized on them are used, a protein can be used as the linker and attached using conventional methods. If microbeads with streptavidin immobilized on them are used, a biotin-modified linker can be used to attach the linker to the microbead. Alternatively, the microbead surface can be chemically modified to introduce a reactive group, and then the linker can be attached by reacting this group with the linker. Chemical modification methods for introducing reactive groups to the microbead surface can be carried out by referring to known methods. The reaction between the microbeads with the introduced reactive group and the linker can also be carried out using conventional methods. For example, if a protein is used as the linker, the amine or carboxyl group present in the protein is reacted with the reactive group (e.g., a carboxyl group or amine) introduced on the bead surface. Methods used in the field of peptide synthesis can also be appropriately utilized. Furthermore, when using sugar chains as linkers, for example, the aldehyde group obtained by oxidizing the sugar chain can be reacted with a reactive group (e.g., an amine) introduced onto the bead surface.

[0043] One embodiment of the microbeads used in the present invention has an anti-human IgGFc antibody bound to its surface. The binding of the anti-human IgGFc antibody to the surface of the microbeads can be carried out by conventional methods. However, it is not limited to this, but for example, by using streptavidin-immobilized microbeads and treating them with biotinylated anti-human IgGFc antibody by conventional methods, microbeads with the anti-human IgGFc antibody immobilized on the surface can be prepared by biotin-avidin binding. Alternatively, by introducing a reactive group that is reactive with proteins (amino acids) to the surface of the microbeads by conventional methods, and then reacting it with the anti-human IgGFc antibody, microbeads with the anti-human IgGFc antibody immobilized on the surface can be prepared. Furthermore, by using protein A or G-immobilized microbeads and treating them with anti-human IgGFc antibody by conventional methods, microbeads with the anti-human IgGFc antibody immobilized on the surface can be prepared.

[0044] In one embodiment of the present invention, the immobilization of an LRRTM molecule and an IgG Fc fusion protein onto the surface of the microbeads can be carried out by an antigen-antibody reaction between an anti-human IgGFc antibody immobilized on the microbead surface and the Fc region of human IgG in the fusion protein. This allows for the preparation of microbeads with LRRTM molecules immobilized on their surface. Figure 1 schematically represents a microbead with two LRRTM molecules immobilized on its surface. In this embodiment, four LRRTM molecules can be immobilized at the same location on the surface of the microbead.

[0045] One aspect of the present invention is a method for inducing presynaptic formation in nerve cells using microbeads on which LRRTM molecules prepared as described above are immobilized. Induction of presynaptic terminal formation (hereinafter sometimes simply referred to as synapse formation induction) can be performed by co-culturing nerve cells with the microbeads of the present invention. Culturing can be carried out by appropriately referring to various reported nerve cell culture conditions. For example, motor nerve cells differentiated from human iPS cells can be cultured and seeded into a matrix-coated 96-well plate. Alternatively, differentiated motor nerve cells can be cultured to create neurospheres, and one neurosphere can be seeded into each well. Preferably, the method using neurospheres is used. After culturing in nerve cell culture medium for a certain period, microbeads with the LRRTM molecule of the present invention immobilized on their surface are added to each well, and cultivation is continued to induce presynaptic terminal formation. There are no particular restrictions on the concentration of microbeads added to each well, and can be appropriately selected according to the experimental system.

[0046] Presynaptic formation can be confirmed by detecting the expression of presynaptic markers. Examples of markers, though not limited to these, include synapsin, synaptophysin, synaptobrevin, neurotransmitter transporters (VAChT, VGlut1, VGlut2, etc.), SNAP25, Bassoon, and Piccolo, but synapsin and synaptophysin are preferred. Marker detection can be performed, for example, using antibodies against each marker and employing immunohistochemistry.

[0047] Confirmation of presynaptic terminal formation can also be performed by detecting neurotransmitters released from the synapse. The neurotransmitters to be detected are not particularly limited and can be arbitrarily selected depending on the purpose. For example, but are not limited to, acetylcholine, amino acids, monoamines, neuropeptides (polypeptides), etc. By detecting and measuring these neurotransmitters, the functionality of synapses, including presynaptic terminals induced from nerve cells using the method of the present invention, can also be evaluated.

[0048] Another aspect of the present invention is a microbead on which the LRRTM molecule prepared as described above is immobilized, which can be used to culture nerve cells and induce synapse formation. The use of such microbeads can be carried out according to the above description.

[0049] Another aspect of the present invention is a method for screening drugs for neurological diseases by using microbeads on which the LRRTM molecule prepared as described above is immobilized, and by adding a target substance to the culture medium and culturing nerve cells differentiated from iPS cells derived from patients suffering from neurological diseases, either during or after induction of presynaptic formation.

[0050] Another aspect of the present invention is a method for screening substances that promote or inhibit neurotransmitter release using synapses induced from nerve cells using microbeads on which LRRTM molecules prepared as described above are immobilized.

[0051] Another aspect of the present invention is a method for diagnosing a subject having a disease by using microbeads on which the LRRTM molecule prepared as described above is immobilized, inducing presynaptic formation using nerve cells differentiated from human-derived iPS cells, and then detecting a specific neurotransmitter released from the synapse into the culture medium or detecting the expression of a protein related to the release of a specific neurotransmitter from the synapse, thereby determining whether the subject is suffering from a specific disease based on the association between a specific neurotransmitter and the disease. [Examples]

[0052] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.

[0053] Cell type and culture medium Human iPS-induced glutamatergic neurons and human iPS-induced motor neurons were purchased from CDI (Cellular Dynamics International, Inc.) (USA). Neurobasal plus medium, B27 plus supplement (Thermo Fischer Scientific) was used as the culture medium, supplemented with 20 μg / ml BDNF, 20 μg / ml GDNF, and penicillin / streptomycin.

[0054] (Example 1) Activated microbeads and control beads (1) Preparation of LRRTM2-Fc-anti-human IgGFc antibody microbeads Streptavidin-immobilized microbeads (Bangs Laboratories, Inc; polystyrene, average diameter 9.94 μm) were washed twice with wash buffer (PBS, 0.01% BSA, 0.05% Triton X-100), and then reacted with biotinylated anti-human IgG (Fc specific) antibody (Sigma-Aldrich; mouse monoclonal) in binding buffer (PBS, 0.01% BSA) to immobilize the biotinylated anti-human IgG (Fc specific) antibody onto the streptavidin-immobilized microbeads. These beads were washed three times with wash buffer to obtain streptavidin-anti-human IgGFc antibody beads.

[0055] Next, streptavidin-anti-human IgGFc antibody beads were suspended in binding buffer. A fusion protein of the extracellular domain of LRRTM2 (amino acids 1 to 389 shown in SEQ ID NO: 2) and the Fc portion of human IgG (LRRTM2-Fc; R&D Systems) was added, and LRRTM2-Fc was immobilized onto the streptavidin-anti-human IgGFc antibody beads. After the reaction, the beads were washed in wash buffer and suspended in binding buffer. This was prepared as the LRRTM2-Fc-anti-human IgGFc antibody microbead suspension.

[0056] (2) Fc-anti-human IgGFc antibody microbeads The protein was prepared in the same manner as described above, by adding the Fc portion of human IgG (Native human IgG Fc fragment protein; Abcam) instead of the fusion protein of the extracellular domain of LRRTM2 and the Fc portion of human IgG.

[0057] (3) LRRTM2-Fc-microbeads Protein A-immobilized microbeads (Bangs Laboratories, Inc; made of polystyrene, average diameter 9.94 μm) were washed twice with wash buffer (PBS, 0.01% BSA, 0.05% Triton X-100), suspended in binding buffer, and then LRRTM2-Fc (R&D Systems) was added to create beads in which the LRRTM2-Fc fusion protein was directly immobilized onto the Protein A-immobilized microbeads. These microbeads were washed three times and then suspended in binding buffer to obtain an LRRTM2-Fc-microbead suspension.

[0058] (4) Fc-microbeads The LRRTM2-Fc fusion protein was prepared using an Fc protein lacking the LRRTM2 portion (Native human IgG Fc fragment protein; Abcam) in the same manner as described in (3) above.

[0059] (Example 2) The activity of LRRTM2 in inducing presynaptic formation in human glutamatergic neurons was investigated as follows. Human iPS-induced glutamatergic neurons were purchased from CDI. Following CDI's protocol, 4 x 10⁶ cells were used. 4 Neurospheres containing individual cells were prepared and cultured for 2-3 days. These neurospheres were then individually seeded into matrix-coated 96-well plates. The neurospheres were cultured for 7-10 days in neuronal medium (Neurobasal plus medium, B27 plus supplement, 20 μg / ml BDNF, 20 μg / ml GDNF, penicillin / streptomycin). Afterward, 0.5 μL of LRRTM2-Fc-anti-human IgGFc antibody microbeads or Fc-anti-human IgGFc antibody microbeads (negative control) suspension was added to each well, and the cells were co-cultured for 20-48 hours. The co-cultured cells were then fixed with 2% PFA, permeabilized with a surfactant, and blocked. Immunostaining with a presynaptic marker (synapsin) was then performed. Simultaneously, immunostaining with betaIII tubulin antibody was also performed to visualize neurites. Immunostaining was performed using the following antibodies: Anti-betaIII tubulin (Tuj1), mouse monoclonal (marker of neurites); Anti-synapsin (Synaptic Systems), rabbit polyclonal (marker of presynaptic cells). Blocking was performed using Blocking Buffer (PBS + 2% Normal Goat Serum + 1% BSA + 0.02% Triton X100).

[0060] As a result, synapsin accumulation was induced at the adhesion site between human iPS-induced glutamatergic nerve axons and LRRTM2-Fc-anti-human IgGFc antibody microbeads, whereas no such accumulation was observed on the negative control Fc-anti-human IgGFc antibody microbeads. The results are shown in Figure 2. This experiment confirmed that the extracellular domain of LRRTM2 has presynaptic formation-inducing activity in human iPS-induced glutamatergic neurons.

[0061] (Example 3) Next, we investigated the relationship between the method of immobilizing LRRTM2 onto microbeads and its presynaptic formation-inducing activity. LRRTM2-Fc-anti-human IgGFc antibody microbeads, Fc-anti-human IgGFc antibody microbeads (negative control), LRRTM2-Fc-microbeads, and Fc-microbeads (negative control) were co-cultured with human iPS-induced glutamatergic neurons, and after immunostaining with synapsin as in Example 2, the presynaptic formation activity was compared. As a result, clear synapsin accumulation was frequently observed at the adhesion sites between human iPS-induced glutamatergic neuronal axons and LRRTM2-Fc-anti-human IgGFc antibody microbeads, while no clear synapsin accumulation was observed at the adhesion sites with LRRTM2-Fc-microbeads. The results of immunostaining are shown in Figure 3. Furthermore, no synapsin accumulation occurred on the respective negative controls, Fc-anti-human IgGFc antibody microbeads or Fc-microbeads. These results suggest that the method of immobilizing LRRTM2-Fc onto microbeads is important for acquiring presynaptic formation-inducing activity in iPS-induced glutamatergic neurons. Furthermore, in the preparation of LRRTM2-Fc-anti-human IgGFc antibody microbeads, when immobilizing LRRTM2-Fc onto the microbeads, if the concentration of LRRTM2-Fc in the added solution was sequentially diluted, i.e., when the amount of LRRTM2-Fc immobilized on the beads was progressively reduced, a significant decrease in activity was observed from a certain point. This indicates that immobilization of LRRTM-2 above a certain level is important, and that the proportion of beads with positive synapsin-inducing activity decreased with decreasing immobilization amount. The results are shown in Figure 4. This suggests that the number of molecules and molecular mobility when LRRTM2-Fc is immobilized onto microbeads may be important for acquiring presynaptic formation-inducing activity.

[0062] (Example 4) Similar studies were conducted on presynaptic formation in human motor neurons. Human iPS-induced motor neurons were purchased from CDI. Following CDI's protocol, 2 x 10⁶ 4Neurospheres containing individual cells were prepared and cultured for 2-3 days. These neurospheres were then individually seeded into matrix-coated 96-well plates. The neurospheres were cultured for 10-21 days in neuronal medium (Neurobasal plus medium, B27 plus supplement, 20 μg / ml BDNF, 20 μg / ml GDNF, penicillin / streptomycin). Afterward, 0.5 μL of LRRTM2-Fc-anti-human IgGFc antibody microbeads or Fc-anti-human IgGFc antibody microbeads (negative control) suspension was added to each well, and the cells were co-cultured for 20-48 hours. Immunostaining was performed in the same manner as in the above experiment, but in addition to synapsin, VAChT (vesicular acetylcholine transporter) was also used as a presynaptic marker. Anti-VAChT (Synaptic Systems), rabbit polyclonal antibody was used.

[0063] As a result, synapsin strongly accumulated at the adhesion site between human iPS-induced motor neuron axons and LRRTM2-Fc-anti-human IgGFc antibody microbeads, while no synapsin accumulation occurred at the adhesion site with the negative control bead, Fc-anti-human IgGFc antibody microbeads. The results are shown in Figure 5. Furthermore, as shown in Figure 6, VAChT accumulation was observed at the adhesion site between human iPS-induced motor neuron axons and LRRTM2-Fc-anti-human IgGFc antibody microbeads, but no accumulation was observed at the adhesion site with the negative control bead. These findings demonstrate that the extracellular domain of LRRTM2 has cholinergic presynaptic formation induction activity in human motor neurons. This fact is an extremely valuable and novel discovery in the previously understated mechanism of synapse formation in motor neurons.

[0064] (Example 5) Similar to Example 3, the relationship between the cholinergic presynaptic formation-inducing activity in motor neurons and the method of immobilizing LRRTM2 onto microbeads was also investigated. LRRTM2-Fc-anti-human IgGFc antibody microbeads, Fc-anti-human IgGFc antibody microbeads (negative control), LRRTM2-Fc-microbeads, and Fc-microbeads (negative control) were co-cultured with human iPS-induced motor neurons. Immunostaining was then performed, and presynaptic formation was compared. As a result, clear accumulation of synapsin and VAChT was frequently observed at the adhesion sites between human iPS-induced motor neuron axons and LRRTM2-Fc-anti-human IgGFc antibody microbeads, while no clear accumulation of synapsin or VAChT was observed at the adhesion sites with LRRTM2-Fc-microbeads. The results are shown in Figure 7. Furthermore, no accumulation of synapsin or VAChT occurred on the respective negative controls, Fc-anti-human IgGFc antibody microbeads and Fc-microbeads. These results suggest that the method of immobilizing LRRTM2-Fc onto microbeads is important for acquiring cholinergic presynaptic formation-inducing activity in iPS-induced motor neurons.

[0065] (Example 6) Synaptic induction from motor neurons derived from healthy individuals and ALS patients was performed as follows. Healthy human iPS-induced motor neurons and ALS patient iPS-induced motor neurons were purchased from iXcells. Presynaptic formation of human motor neurons was performed in the same manner as in Example 4. However, since the iPS-induced motor neurons in this example were purchased from iXcells, the creation of neurospheres followed iXcells' protocol. Immunostaining was performed to identify the neurotransmitters contained in the presynaptic cells induced as described above. Synapsin was used as a marker for all presynaptic cells, and all presynaptic cells were detected. On the other hand, VAChT was used as a marker for cholinergic presynaptic cells, and synaptic vesicles accumulating acetylcholine as a neurotransmitter were detected. As a result, synapsin-positive presynaptic cells were induced to a similar extent from motor neurons of healthy individuals and ALS patients, but the positivity rate of VAChT in presynaptic cells induced from motor neurons of ALS patients was significantly lower than that of motor neurons of healthy individuals (Figure 8). This suggests that motor neurons derived from ALS patients have a low capacity to release acetylcholine. Therefore, it is suggested that it is possible to distinguish between motor neurons of healthy individuals and those of ALS patients by detecting the amount of acetylcholine released in response to neuronal stimulation.

[0066] (Example 7) Using synapses induced from motor neurons of ALS patients and synapses induced from motor neurons of healthy individuals, prepared by the method of Example 6, the amount of acetylcholine released from the synapses can be compared as follows. Glutamate is added as an acetylcholine release inducer to a culture medium containing the cell bodies of motor neurons in which synapses have been induced, thereby inducing acetylcholine release. After culturing for several days, the medium is collected, and the amount of acetylcholine in the medium is measured by ELISA, allowing for a comparison of the acetylcholine release capacity of synapses derived from ALS patients and synapses derived from healthy individuals.

[0067] (Example 8) Using the method of the present invention, we screened for compounds that affect synapse formation. Using 1814 compounds listed in the FDA-approved drug compound library, synapsin formation was confirmed in the same manner as in Example 2. Each compound to be evaluated was added together with microbeads to a final concentration of 10 μM. Immunostaining of the presynapsin marker (synapsin 1) was performed, and the fluorescence intensity on the microbeads was measured. Seven compounds showed a strictly standardized mean difference (SSMD) of 2 or higher, confirming that they significantly promoted synapse formation.

[0068] The above detailed description merely illustrates the object and subject matter of the present invention and does not limit the scope of the appended claims. Various modifications and substitutions to the embodiments described without departing from the scope of the appended claims will be apparent to those skilled in the art from the teachings described herein. [Industrial applicability]

[0069] The method and microbeads of the present invention are useful as tools for inducing synapse formation in nerve cells and can also be used in screening methods.

Claims

1. A method for screening drugs for neurological diseases, comprising the following steps: (i) A step of co-culturing motor neurons differentiated from iPS cells derived from patients suffering from neurological diseases with microbeads having an average diameter of 5 to 20 μm on which a fusion protein containing the LRRTM2 molecule is immobilized on its surface to induce the formation of a presynaptic terminal, wherein the fusion protein containing the LRRTM2 molecule comprises at least an extracellular domain of the LRRTM2 molecule and the Fc region of human IgG, the microbeads have an anti-human IgGFc antibody immobilized on their surface, and the fusion protein containing the LRRTM2 molecule is immobilized on the surface of the microbeads via binding between the anti-human IgGFc antibody immobilized on the surface of the microbeads and the Fc region of human IgG of the fusion protein containing the LRRTM2 molecule. (ii) A step of adding the target substance to the culture medium and culturing it in step (i) or after step (i), and (iii) Next, a step to detect the effect of the target substance on the formation of the presynaptic terminal in the motor nerve cell, wherein the detection is performed by detecting the expression of proteins related to the release of neurotransmitters from the synapse. A screening method that includes [this].

2. The screening method according to claim 1, wherein the detection of the expression of a protein related to the release of the neurotransmitter from the synapse is performed by detection of fluorescence by immunostaining.

3. A screening method that performs the method described in claim 1 or 2 using high-throughput screening (HTS).

4. A method for screening substances that promote or inhibit the release of neurotransmitters, comprising the following steps: (a) A step of co-culturing human motor neurons with microbeads having an average diameter of 5 to 20 μm on which a fusion protein containing an LRRTM2 molecule is immobilized on its surface to induce presynaptic formation, wherein the fusion protein containing the LRRTM2 molecule comprises at least an extracellular domain of the LRRTM2 molecule and the Fc region of human IgG, the microbeads have an anti-human IgGFc antibody immobilized on their surface, and the fusion protein containing the LRRTM2 molecule is immobilized on the surface of the microbeads via binding between the anti-human IgGFc antibody immobilized on the surface of the microbeads and the Fc region of human IgG of the fusion protein containing the LRRTM2 molecule. (b) Next, the motor nerve cells are cultured in a culture medium in which the target substance has been added, and (c) A step of detecting the expression of proteins related to the release of neurotransmitters from synapses using fluorescence by immunostaining, A screening method that includes [this].

5. The screening method according to claim 4, further comprising adding a substance that stimulates synapses and induces the release of neurotransmitters to the culture medium in step (b).

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