Method for producing a material for treating brain damage, method for treating brain damage, method for producing a material for regenerating brain nerve cells, and method for regenerating brain nerve cells

Abstract

To provide a material for treating encephalopathy, a method for treating encephalopathy, a material for regenerating brain neuron, and a method for regenerating brain neuron.MEANS: The present invention provides a material for treating encephalopathy or the like, containing a support fixed or coated with at least one selected from the group consisting of N-cadherin, a fusion protein containing the whole or some parts of N-cadherin, and a fusion protein containing the whole or some parts of a protein having homology with N-cadherin.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a material for treating brain damage, a method for treating brain damage, a material for regenerating brain nerve cells, and a method for regenerating brain nerve cells. [Background technology]

[0002] Neonatal brain damage, such as hypoxia-ischemia, is a leading cause of infant death and lifelong disability. However, there are currently no therapeutic methods to repair damaged brain tissue. The "ventricular-subventricular zone (V-SVZ)" is a neural stem cell (NSC) niche in the postnatal vertebrate brain, providing a continuous supply of newborn neurons (Kaneko et al., 2014). Notably, the human neonatal V-SVZ has an extraordinary capacity for neuronal production (Paredes et al., 2016; Sanai et al., 2011), suggesting that the V-SVZ is likely a source of endogenous neural regeneration after neonatal brain injury.

[0003] In rodents, newborn neurons use various scaffolds for migration. In injured adult brains, newborn neurons from the V-SVZ migrate toward the lesion along blood vessels (Yamashita et al., 2006). Transplantation of vascular-mimicking scaffolds into injured adult brains promotes the migration of newborn neurons toward the lesion (Ajioka et al., 2015; Fujioka et al., 2017). Compared with adult brains, more newborn neurons migrate from the V-SVZ toward the lesion in neonatal brains (Covey et al., 2010). However, the role of neonatal scaffolds in guiding newborn neurons toward the lesion has not yet been thoroughly studied.

[0004] Radial glia (RG) are cells whose cell bodies reside in the ventricular zone and extend thin processes to the pial surface. They function as neural stem cells (NSCs) during the fetal period (Rakic, 1972). In the immature cerebral cortex, newborn neurons use radial glia as a scaffold for migration (Kawauchi et al., 2010). During this process, radial glial processes form adherens junction (AJ)-like structures with newborn neurons, guiding them appropriately for the formation of cortical layers (Franco et al., 2011; Rakic, 1972). Shortly after birth, radial glia transform into astrocytes or ependymal cells (Kriegstein and Alvarez-Buylla, 2009). Therefore, it was unknown how migratory newborn neurons are induced after neonatal brain injury. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kaneko et al., 2017, J. Neurochem. 141, 835-847. [Non-patent document 2] Paredes et al., 2016, Science 354, aaf7073. [Non-patent document 3] Sanai et al., 2011, Nature 478, 382-386. [Non-patent document 4] Yamashita et al., 2006, J. Neurosci. 26, 6627-6636. [Non-Patent Document 5] Ajioka et al., 2015, Tissue Eng. Part A 21, 193-201. [Non-patent document 6] Fujioka et al., 2017, EBioMedicine 16, 195-203. [Non-Patent Document 7] Covey et al., 2010, Dev. Neurosci. 32, 488-498. [Non-patent document 8] Rakic, 1972, J. Comp. Neurol. 145, 61-83. [Non-Patent Document 9] Kawauchi et al., 2010, Neuron 67, 588-602. [Non-Patent Document 10] Franco et al., 2011, Neuron 69, 482-497. [Non-Patent Document 11] Kriegstein and Alvarez-Buylla, 2009, Annu. Rev. Neurosci. 32, 149-184. Summary of the Invention [Problem to be solved by the invention]

[0006] Despite the above findings, there is currently no method to treat brain disorders.

[0007] Therefore, an object of the present invention is to provide a material for treating brain damage, a method for treating brain damage, a material for regenerating brain nerve cells, and a method for regenerating brain nerve cells. [Means for solving the problem]

[0008] Although radial glia normally disappear shortly after birth, the present inventors have discovered that radial glial processes can persist in the brains of injured neonatal mice and serve as a scaffold for postnatal migration of newly generated neurons derived from the ventricular-subventricular zone (V-SVZ) to the injured area. This injury-induced persistence of radial glial processes occurs only for a limited time after birth, and promotes the directional saltatory movement of newly generated neurons through cell-cell adhesion mediated by N-cadherin, which promotes RhoA activity. Based on these findings, the present inventors further investigated the possibility of transplanting an N-cadherin-containing scaffold into the injured neonatal brain, which similarly promotes the migration and maturation of newly generated neurons derived from the V-SVZ, improving the function of impaired locomotor behavior, and thereby solving the above-mentioned problems. This finding led to the completion of the present invention.

[0009] That is, the present invention relates to the following [1] to

[15] related to a material for treating brain damage, a method for treating brain damage, a material for regenerating brain nerve cells, and a method for regenerating brain nerve cells. [1] A material for treating brain disorders, characterized by comprising a carrier onto which one or more members selected from the group consisting of N-cadherin, a fusion protein containing all or a partial region of N-cadherin, and a fusion protein containing all or a partial region of a protein having homology to N-cadherin are immobilized or coated. [2] A material for treating brain disorders according to [1] above, wherein the fusion protein containing all or a portion of the N-cadherin region and the fusion protein containing all or a portion of a protein homologous to N-cadherin have the ability to bind with homophilicity to N-cadherin. [3] A material for treating brain disorders according to [1] or [2], wherein the fusion protein comprising all or a partial region of N-cadherin and the fusion protein comprising all or a partial region of a protein homologous to N-cadherin are fusion proteins comprising a protein selected from the following (1) to (3): (1) N-cadherin or a protein whose amino acid sequence is 90% or more identical to N-cadherin. (2) The extracellular domain of N-cadherin, or a protein whose amino acid sequence is 90% or more identical to the extracellular domain of N-cadherin. (3) A protein containing one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain, and EC5 domain of N-cadherin. [4] The material for treating a brain disorder according to any one of [1] to [3] above, wherein the fusion protein is a fusion protein with the Fc region of immunoglobulin. [5] The material for treating a brain disorder according to any one of [1] to [4] above, wherein the carrier is a porous body. [6] The material for treating brain disorders according to any one of [1] to [5] above, wherein the carrier is a biomaterial or a biocompatible polymer carrier. [7] The material for treating a brain disorder according to any one of [1] to [6] above, wherein the carrier is a porous body of a biomaterial. [8] The material for treating brain disorders according to [6] or [7] above, wherein the biomaterial is a protein or a polysaccharide. [9] The material for treating a brain disorder according to any one of [6] to [8] above, wherein the biomaterial is gelatin or collagen.

[10] The material for treating brain disorders according to any one of [1] to [9] above, wherein the carrier is a gelatin sponge.

[11] A material for regenerating brain neurons, characterized by comprising a carrier on which one or more members selected from the group consisting of N-cadherin, a fusion protein containing all or a partial region of N-cadherin, and a fusion protein containing all or a partial region of a protein having homology to N-cadherin are immobilized or coated.

[12] A method for treating a brain disorder, comprising transplanting the material for treating a brain disorder according to any one of the above [1] to

[10] into the brain.

[13] The method for treating a brain disorder according to

[12] above, characterized in that neural cells derived from pluripotent stem cells are transplanted into the brain simultaneously with the therapeutic material or after the therapeutic material has been transplanted into the brain.

[14] A method for regenerating brain nerve cells, comprising transplanting the material for regenerating brain nerve cells according to

[11] above into the brain.

[15] The method for regenerating brain neurons according to

[14] above, characterized in that neural cells derived from pluripotent stem cells are transplanted into the brain simultaneously with the regenerative material or after the regenerative material has been transplanted into the brain. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a material for treating brain damage, a method for treating brain damage, a material for regenerating brain nerve cells, and a method for regenerating brain nerve cells. [Brief explanation of the drawings]

[0011] [Figure 1]Radial glia maintain their processes after neonatal brain injury and provide a scaffold for the migration of newborn neurons from the V-SVZ. (A) Experimental scheme. (B) Coronal sections of the cortex from Dcx-EGFP mice at 7 dpi stained for GFP (green) and nestin (red). Arrowheads indicate GFP-positive newborn neurons associated with nestin-positive processes (B1-B4). (C) Coronal sections of the cortex from wild-type (WT) mice. A plasmid expressing EmGFP was electroporated into the V-SVZ, and the sections were stained for GFP (green), Dcx (red), and nestin (white). (D) Dcx-positive (green) newborn neurons (asterisks) and nestin-positive (white) radial glial processes (arrows) express N-cadherin (red). (E) Neonatal radial glial processes after injury following adenovirus infection. Coronal sections of the cortex from R26-tdTomato mice stained for DsRed (red) and nestin (white). Yellow and white arrows indicate radial glial processes in the V-SVZ and corpus callosum (CC), respectively (E'). (F-J) Effects of DN-N-cadherin (F-H) or N-cadherin-KD (I-J) expression in radial glial processes on the adhesion of newborn neurons to radial glial processes (F, G-I) and their migration toward the lesion (F, H-J). Coronal sections of the cortex from R26-tdTomato;Dcx-EGFP mice stained for GFP (green), DsRed (red), and nestin (white) (F). (G-I) Percentage of the total amount of newborn neurons along radial glial processes (Figure S2F shows the entire cell body contact). (K and L) TEM images of newborn neurons (N, green) and control (K) and DN-N-cadherin-expressing (L) radial glial processes (RGF, red). Red arrows and blue arrowheads indicate AJ-like electron-dense structures and irregular contacts, respectively. (M) Contact density and ratio of irregular contact areas at the contact points between newborn neurons and radial glial processes. Scale bars are 10 μm (B), 50 μm (E), 5 μm (C, D, and F), and 500 nm (K and L). Error bars indicate ± standard error of the mean (± SEM). [Figure 2]N-cadherin promotes RhoA activity and saltatory motility of newborn neurons migrating along radial glial processes. (A) Time-lapse images at 5 dpi of GFP-positive newborn neurons (green) migrating along control radial glial processes and DN-N-cadherin-expressing tdTomato-positive processes (purple) in injured cortical slices. Arrows and arrowheads indicate the leading tip of the newborn neuron and the radial glial process, respectively. (B-G) Newborn neuron migration speed (B), percentage of time spent in the radial glial process adhesion phase (C), percentage of newborn neurons not attached to radial glial processes (D), stride length (E), percentage of time spent in the resting phase (F), and migration cycle time (G). (H and I) Time-lapse FRET ratiometric images of RhoA activity (pseudocolor) in cultured newborn neurons (H). A magnified image is shown in (I). (J) RhoA activation. (KP) Migratory behavior of cultured newborn neurons on N-cadherin-Fc stripes. (K) Time-lapse images of tdTomato-positive newborn neurons (red). Newborn neurons show migration speed (L), percentage of time spent in the resting phase (M), stride length (N), and migration cycle time (O). (P) Preference for N-cadherin-Fc stripes. Dashed lines (H and K) indicate stripe boundaries. Scale bar: 10 μm. Error bars indicate ± standard error (± SEM). [Figure 3]N-cadherin-containing scaffolds promote the migration and maturation of V-SVZ-derived newborn neurons after neonatal brain injury. (A and B) Coronal sections of the cortex from control (A) and DN-N-cadherin (B) groups stained for EmGFP (green). These are composites of eight separate fields (two vertical and four horizontal tiles). (C) Number of EmGFP·NeuN double-positive cells in the injured cortex. (D) Time-lapse images of cultured newborn neurons migrating along control and N-cadherin sponges (Sp). (E) Velocity of cultured newborn neurons. (F) Experimental scheme. (G) EmGFP-positive (green) V-SVZ-derived Dcx-positive (red) newborn neurons within an N-cadherin sponge (orange). (H) Coronal sections of the cortex from sponge-treated wild-type mice (P2, P14, and 8w models) stained for Dcx (red). Arrows indicate Dcx-positive cells along the sponge. (I) Density of Dcx-positive cells within the sponge. (J and J') Coronal sections of the cortex from a P30 wild-type mouse implanted with a sponge, stained for EmGFP (green) and NeuN (red). Arrows indicate EmGFP·NeuN double-positive neurons. (K) Number (left) and distribution (right) of EmGFP·NeuN double-positive neurons in the injured cortex. Scale bars are 50 μm (A, B, H, and J) and 10 μm (D and G). Error bars indicate ± standard error of the mean (± SEM). [Figure 4] N-cadherin-containing scaffolds improve functional recovery by promoting V-SVZ-derived neuronal regeneration after neonatal brain injury. (A-C) Catwalk analysis at P30. Forepaw "maximum contact area" (A), "print area (total area of ​​footprint)" (B), and "base of support (distance between left and right forelimbs)" (C). (D) Foot-fault test. Percentage of left foot-faults in the P2, P14, and 8w injury models. (E) Experimental scheme. (F) Strategy for excluding V-SVZ-derived newborn neurons. (G) Number of EmGFP·NeuN double-positive newborn neurons in the injured cortex at P30. (H) Foot-fault test in Ad-Cre;NSE-DTA mice implanted with N-cadherin sponges. Error bars indicate ± standard error (± SEM). DETAILED DESCRIPTION OF THE INVENTION

[0012] The material for treating brain injury of the present invention can promote the migration and maturation of migratory newborn neurons, particularly newborn neurons derived from the V-SVZ (ventricular-subventricular zone), to affected areas such as damaged areas. By transplanting the material for treating brain injury of the present invention into the brain, motor function impaired by brain injury, for example, can be improved.

[0013] The material for regenerating brain nerve cells of the present invention can promote the migration and maturation of migratory new neurons, particularly new neurons derived from the V-SVZ (ventricular-subventricular zone), to affected areas such as damaged areas, thereby increasing the number of nerve cells.

[0014] The material for treating brain damage, the method for treating brain damage, the material for regenerating brain nerve cells, and the method for regenerating brain nerve cells of the present invention will be described in detail below.

[0015] [Materials for treating brain disorders and regenerating brain nerve cells] The materials for treating brain disorders and for regenerating brain neurons of the present invention are characterized by being carriers onto which one or more members selected from the group consisting of N-cadherin, fusion proteins containing all or a portion of a region of N-cadherin, and fusion proteins containing all or a portion of a region of a protein having homology to N-cadherin (hereinafter also abbreviated as "N-cadherin, etc.") are immobilized or coated.

[0016] Cadherins are Ca receptors that bind to the nuclei of cells called adherens junctions. 2+Cadherins are adhesion molecules involved in cell-cell adhesion and junctions dependent on the cellular context, and three types are known: E (epithelial), N (neuronal), and P (placental). These cadherin molecules are membrane-bound glycoprotein molecules consisting of 700-750 amino acid residues, and their extracellular regions contain five repeating structures of approximately 110 amino acid residues, known as extracellular cadherin (EC) domains. For example, in the case of human N-cadherin (the amino acid sequence of which is shown in SEQ ID NO: 1), the EC1, EC2, EC3, EC4, and EC5 domains correspond to residues 160-267, 268-382, 383-497, 498-603, and 604-714, respectively (the numbers represent the residue numbers in the amino acid sequence shown in SEQ ID NO: 1). In the case of mouse N-cadherin (the amino acid sequence of which is shown in SEQ ID NO: 2), the EC1, EC2, EC3, EC4, and EC5 domains correspond to residues 160-267, 268-382, 383-497, 498-603, and 604-717, respectively (the numbers represent the residue numbers in the amino acid sequence shown in SEQ ID NO: 2). These EC domains share homology among different cadherin molecular species, with the N-terminal domains (EC1 and EC2) being particularly homologous.

[0017] N-cadherin is a calcium-dependent cell adhesion molecule (CCM) protein of approximately 140 kD. N-cadherin plays an important role in cell adhesion through interactions with homologous cadherins and catenin-mediated binding to the actin cytoskeleton, and is involved in developmental differentiation. N-cadherin is expressed in various tissues, including nerves, cardiac muscle, skeletal muscle, and vascular endothelium. N-cadherin has been reported to function as a key regulator of nervous system development by providing important molecular signals in many developmental processes, such as retinal development, somitogenesis, and neurite outgrowth (Miyataniet al., Science 1989;245;631-5; Hansen et al., Cell Mol. Life Sci. 2008:65;3809-21).

[0018] The method for producing N-cadherin and the fusion protein is not particularly limited, but it is preferable to produce and purify recombinant proteins using molecular biological techniques and use them. Any other method that produces a similar effect can also be used. For example, N-cadherin can be extracted from biological tissues or cells, purified, or chemically synthesized.

[0019] Standard protocols for producing recombinant N-cadherin and the fusion proteins and for obtaining genes encoding these molecules have already been established, and practitioners can refer to the reference books listed above, but this is not intended to be limiting. The N-cadherin gene has already been isolated and identified in animals such as humans and mice, and its nucleotide sequence is available in public DNA databases such as NCBI (NCBI accession numbers: human NM_001792, mouse NM_M31131, M22556, etc.). Therefore, those skilled in the art can design primers or probes specific to the N-cadherin gene and obtain and use cDNA for the N-cadherin gene using standard molecular biology techniques. N-cadherin cDNA can also be purchased from OriGene Technologies, Inc. [https: / / www.origene.com / ] and other sources. The gene used is preferably derived from an animal of the same species as the target of treatment. However, genes derived from different animal species may also be used.

[0020] A suitable method for producing recombinant N-cadherin and fusion proteins involves introducing and expressing a gene encoding the molecule in mammalian cells such as COS cells, 293 cells, or CHO cells. Preferably, the gene is linked to a nucleic acid sequence, i.e., a promoter sequence, that enables gene transcription and expression in a wide range of mammalian cells, in a manner that allows transcription and expression under the control of the promoter. Furthermore, the gene to be transcribed and expressed is preferably linked to a poly(A) addition signal. Suitable promoters include promoters derived from viruses such as Simian Virus (SV) 40, cytomegalovirus (CMV), and Rous sarcoma virus, as well as the β-actin promoter and the EF (Elongation Factor) 1α promoter.

[0021] The gene used to produce the recombinant protein does not need to contain the entire gene encoding the molecule; even a partial gene sequence can be used, as long as the protein or peptide molecule encoded by the partial sequence has adhesive activity equivalent to or greater than that of the original molecule. For example, a protein containing the EC1-EC5 domains encoding the extracellular region can be used. Furthermore, since the N-terminal domain (EC1) of cadherin molecules generally determines the binding specificity, i.e., homophilicity, of the molecule (Nose et al., Cell 61:147, 1990), it is also possible to produce and use a protein molecule that contains at least EC1 but lacks one or more of the other domains.

[0022] It is preferable that the fusion protein containing all or a portion of the N-cadherin region and the fusion protein containing all or a portion of the protein having homology to N-cadherin have the ability to bind to N-cadherin with homophilicity.

[0023] Furthermore, it is preferable that the fusion protein containing all or a portion of the N-cadherin region and the fusion protein containing all or a portion of a protein homologous to N-cadherin are fusion proteins containing a protein selected from the following (1) to (3): (1) N-cadherin or a protein having an amino acid sequence that is 80% or more (preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more) identical to that of N-cadherin. (2) The extracellular domain of N-cadherin, or a protein whose amino acid sequence is 80% or more (preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more) identical to the extracellular domain of N-cadherin. (3) A protein containing one or more of the EC1 domain, EC2 domain, EC3 domain, EC4 domain, and EC5 domain of N-cadherin.

[0024] The fusion protein may be a fusion protein with another protein or peptide, such as an Fc region of immunoglobulin, GST (Glutathione-S-Transferase) protein, MBP (Mannose-Binding Protein), avidin protein, His (oligohistidine) tag, HA (HemAgglutinin) tag, Myc tag, or VSV-G (Vesicular Stromatitis Virus Glycoprotein) tag. Recombinant proteins can be easily and efficiently purified using a protein A / G column or a specific antibody column. Fc fusion proteins are particularly suitable for carrying out the present invention because they exhibit excellent adsorption to biomaterials, including synthetic polymers such as polylactic acid and polydioxane, biomaterials such as collagen, ceramics such as apatite, and metals such as titanium alloys and stainless steel.

[0025] Numerous genes encoding the Fc region of immunoglobulins have already been isolated and identified in mammals, including humans. Numerous nucleotide sequences have also been reported. For example, sequence information for the nucleotide sequences containing the Fc regions of human IgG1, IgG2, IgG3, and IgG4 is available in public DNA databases such as NCBI, and is registered under accession numbers AJ294730, AJ294731, AJ294732, and AJ294733, respectively. Therefore, those skilled in the art can design primers or probes specific to the Fc region and obtain and use cDNA encoding the Fc region portion using standard molecular biology techniques. In this case, the gene encoding the Fc region to be used is not particularly limited to the animal species or subtype, but is preferably a gene encoding the Fc region of human IgG1 or IgG2, or mouse IgG2a or IgG2b, which have strong binding properties to Protein A / G. Furthermore, a method for increasing binding to Protein A by introducing a mutation into the Fc region is also known (see Nagaoka et al., Protein Eng. 16:243, 2003 (Non-Patent Document 7)), and Fc proteins genetically modified using this method can also be used.

[0026] An example of a method for producing such recombinant proteins is reported in a previously published paper (Yue XS et al., Biomaterials 2010; 31:5287-96).

[0027] In addition, a purified recombinant protein (Recombinant Human N-Cadherin Fc Chimera: R&D Systems) is commercially available, which is produced by introducing a fusion gene into mouse cells in which cDNA encoding the extracellular domain of human N-cadherin is linked to a cDNA encoding the Fc domain of human IgG and a His tag sequence, and then expressing the fusion gene. Another commercially available recombinant protein (Recombinant Mouse N-Cadherin Fc Chimera: R&D Systems) is produced by introducing a fusion gene into mouse cells in which cDNA encoding the extracellular domain of mouse N-cadherin is linked to a cDNA encoding the Fc domain of mouse IgG, and then expressing the fusion gene.

[0028] The carrier is preferably a biomaterial or a biocompatible polymer. Biomaterials are not particularly limited, but include proteins and polysaccharides. Proteins include gelatin, collagen, etc. Polysaccharides include chitosan, chitin, etc. Biocompatible polymers include polyethylene glycol, polylactic acid, etc. Furthermore, materials used in injectable gels may also be used as the carrier.

[0029] The shape and properties of the carrier are not particularly limited, but examples thereof include fiber, gel, and porous bodies, with porous bodies being particularly preferred. The porous body is preferably a sponge, as nerve cells migrate through the pores.

[0030] The method for producing the carrier is not particularly limited, and a porous body can be obtained by, for example, freeze-drying a gel biomaterial, and a gelatin sponge can be obtained by freeze-drying gelatin.

[0031] An example of a method for producing a gelatin sponge is a paper previously reported by the present inventors (Ajioka et al., Tissue Eng. Part A 21, 193-201).

[0032] The size of the porous body is not particularly limited, but it is desirable that the size be such that it can be transplanted into the damaged area of ​​the brain.

[0033] The shape of the porous body is not particularly limited, but a shape that allows nerve cells to migrate is desirable.

[0034] The method for immobilizing or coating N-cadherin or the like on a carrier is not particularly limited. Physical methods such as adsorption and chemical methods such as covalent bonding can be applied, but adsorption methods are preferred due to ease of operation. When the adhesive molecule is a proteinaceous or peptide molecule, or a polymer compound containing a sugar chain, the molecule can be easily adsorbed by contacting the carrier with a solution of the molecule and removing the solvent after a certain period of time. More specifically, for example, when the carrier is porous, a solution of the adhesive molecule in a solvent such as distilled water or PBS is filtered and sterilized, and then contacted with a porous material such as a gelatin sponge and left for several hours to overnight to obtain a porous material on which the adhesive molecule is immobilized or coated. Preferably, the porous material is washed several times with distilled water or PBS and then replaced with a balanced salt solution such as PBS before use.

[0035] Furthermore, if an antigenic molecule is artificially attached or fused to the adhesive molecule in advance, binding with a specific antibody to the antigenic molecule can be utilized, which is more preferable because it allows for efficient modification of the adhesive molecule to the substrate surface. In this case, the specific antibody must be immobilized or coated on the support in advance by physical methods such as adsorption or chemical methods such as covalent bonding. For example, in the case of a recombinant protein in which the adhesive molecule is fused to an IgG Fc region protein, an antibody that specifically recognizes the IgG Fc region can be used to modify the support in advance. In the case of a recombinant protein in which the adhesive molecule is fused to various proteins or tag sequence peptides, the support can be used by modifying it in advance with an antibody specific to the fused molecule.

[0036] In carrying out the present invention, two or more proteins selected from the group consisting of N-cadherin, fusion proteins containing all or a partial region of N-cadherin, and fusion proteins containing all or a partial region of a protein having homology to N-cadherin may be used in combination. In this case, solutions of the respective proteins may be mixed, and the resulting mixture may be modified according to the method described above.

[0037] The concentration of the solution of the above-mentioned N-cadherin or the like must be determined appropriately depending on the amount of adsorption and / or affinity of the protein, as well as the physical properties of the protein, but is in the range of approximately 0.01 to 1000 μg / mL, preferably approximately 0.1 to 200 μg / mL, more preferably 1 to 50 μg / mL, and most preferably 3 to 20 μg / mL.

[0038] The brain disorders to be treated with the material for treating brain disorders of the present invention are not particularly limited, and examples thereof include hypoxic encephalopathy, hypoxic-ischemic encephalopathy, ischemic brain disorders, and brain disorders caused by physical injuries.

[0039] [Method for treating brain disorders and regenerating brain nerve cells] The method for treating brain disorders and regenerating brain neurons of the present invention involves transplanting the material of the present invention into the brain. While the transplantation method is not particularly limited, the material of the present invention may be transplanted so as to connect the area where newborn neurons reside with the affected area, allowing neural cells, preferably those derived from the ventricular-subventricular zone (VSVZ), to migrate to the affected area, such as a lesion. Furthermore, by transplanting the material of the present invention into the brain beforehand or simultaneously with neural cells derived from pluripotent stem cells, the material of the present invention can serve as a scaffold for the transplanted neural cells, promoting the migration of neural cells and the regeneration of the affected area. However, transplanting neural cells derived from pluripotent stem cells into the brain before the material is not preferable, as it makes it difficult to efficiently migrate the cells to the damaged area. Examples of pluripotent stem cells include ES cells, ntES cells, and iPS cells. Examples of neural cells include neural stem cells, neural progenitor cells, neurons (nerve cells), and glial cells.

[0040] The amount of the material to be transplanted may be an effective amount depending on the size of the damaged area, etc.

[0041] The material can be implanted into the brain by conventional surgical procedures, such as by exposing the brain through an incision and then implanting the material into the affected area. If the material is an injectable material such as an injectable gel, it can be locally injected into the affected area.

[0042] Furthermore, when the material is transplanted into the brain, it is desirable to do so in a clean state.

[0043] The transplant target may be any patient with a brain disorder, and is not limited to humans, but may also be any other animal, such as a mammal, bird, reptile, amphibian, or fish. [Example]

[0044] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following, "%" is based on mass unless otherwise specified.

[0045] <Experimental animals> All experiments involving live animals were conducted in accordance with the guidelines and regulations of Nagoya City University and approved by the President of Nagoya City University. Animals were housed in a specific pathogen-free facility in a controlled environment (23 ± 1°C, 12-hour light-dark cycle with a shift at exactly 8:00 AM) in cages lined with chip-like bedding and with free access to water and food (MF, Oriental Yeast Co., Ltd.). Wild-type (WT) ICR and C57BL6 / J mice were purchased from SLC Japan. The following transgenic mouse strains were used: We used R26-tdTomato mice (stock no. 7914, Jackson Laboratory), Neurog2-d4Venus mice (Kawaue et al., 2014, Dev. Growth Differ. 56, 293-304), NSE-DTA mice (Imayoshi et al., 2008, Nat. Neurosci. 11, 1153-1161; Kobayakawa et al., 2007, Nature 450, 503-508), and Dcx-EGFP mice (Gong et al., 2003, Nature 425, 917-925) (MMRRC_000244). The R26-tdTomato and NSE-DTA strains are grown on a C57BL6 / J genetic background. The Dcx-EGFP mouse strain was intercrossed with the R26-tdTomato reporter mouse strain (syngeneic). Genotype was confirmed by PCR after tail clipping of the mice. In the R26-tdTomato strain, Cre-mediated recombination of a lox-stop-lox cassette with adenoviral vectors (Ad-CMV-Cre and Ad-CMV-DN-N-cadherin-IRES-Cre) led to ubiquitous, permanent expression of tdTomato. In the NSE-DTA strain, Cre-mediated recombination of a lox-stop-lox cassette with Ad-CMV-Cre led to permanent expression of DTA under the control of the NSE gene promoter, resulting in the ablation of neuronal progeny. Mice were age-matched for each experiment. Before weaning, littermates were housed with their dams or foster dams. After weaning, they were separated by sex and housed in groups (up to seven per cage).In experiments using adult mice, healthy 8-week-old male mice were used. In other animal experiments, both healthy male and female mice were used. Siblings were randomly assigned to test groups.

[0046] <Culture of V-SVZ cells> The neonatal V-SVZ was excised from WT ICR P0-1 pups and dissociated with trypsin-EDTA (Invitrogen). Both male and female pups were used. The cells were washed twice with L-15 medium (GIBCO) containing 40 μg / mL DNaseI (Roche), and then 2 μg of plasmid DNA was introduced using the Amaxa Nucleofector II system (Lonza). The introduced cells were suspended in RPMI-1640 medium (Wako), incubated at 37 °C for 15 minutes to aggregate, and then the aggregates were cut into blocks (150 - 200 μm in diameter), mixed with 50% Matrigel (BD Biosciences) in L-15 medium, and fixed on dishes. The dishes were stored in a humidified incubator at 37 °C and 5% CO2. The gels containing the aggregates were cultured in serum-free Neurobasal medium (GIBCO) containing 2% B-27 supernatant (Invitrogen), 2 mM L-glutamine (GIBCO), and 50 U / mL penicillin-streptomycin (GIBCO) for 48 hours.

[0047] <Brain injury> On postnatal day 2 (P2), P4, P14, and 8-week-old mice were subjected to cryogenic cerebral cortex injury by a known method (Ajioka et al., 2015, Tissue Eng. Part A 21, 193 - 201). Briefly, the mice were deeply anesthetized by natural inhalation of isoflurane, and the skull was exposed by scalp incision. In each of the P2 and P4, P14, and 8-week-old mice, a metal probe (1.5 mm in diameter) cooled with liquid nitrogen was placed stereotactically on the right skull for 30, 60, and 120 seconds (0.5 mm in front of bregma and 1.2 mm lateral). The scalp was immediately sutured, and the mice were returned to the breeding cage. This procedure reproducibly produced lesions with a depth of 500 - 600 μm.

[0048] Hypoxic-ischemic injury was induced in P5 mice. During surgery, mice were deeply anesthetized with natural inhalation of isoflurane. After cauterization of the right common carotid artery under a dissecting microscope, mice were placed in a plastic box at 37°C in a humid environment under systemic hypoxia (oxygen / nitrogen, 8 / 92%) for 20 minutes. After this procedure, mice were returned to their cages.

[0049] <Adenoviral vector and RNAi construct> To generate pENTR4-DN-N-cadherin-IRES-Cre, the IRES-Cre fragment from pLV-CMV-tdTomato-IRES-Cre (Robel et al., 2011, J. Neurosci. 31, 12471-12482) and the DN-N-cadherin fragment from pCAG-MCS2-DN-N-cadherin (Nuriya and Huganir, 2006, J. Neurochem. 97, 652-661) were amplified by PCR and inserted into the BamHI and SalI sites, respectively, of pENTR4-H1 (RIKEN). For N-cadherin knockdown (KD) experiments using adenoviral vectors, the target sequence of the mouse N-cadherin gene was inserted into a modified Block-iT Pol II miR RNAi expression vector (Invitrogen) containing EmGFP. As a control, a lacZ target sequence was used as previously described (Ota et al., 2014, Nat. Commun. 5, 4532). To generate pENTR-tdTomato-miR-lacZ and -N-cadherin, the EmGFP-encoding fragment in the pENTR-EmGFP-RfA plasmid was ligated between the BspMI sites and a PCR-amplified tdTomato fragment from ptdTomato-N1 (Clontech Laboratories) was inserted. The Gateway system (Invitrogen) was used to generate the following adenoviral vectors: pAd-CMV-DN-N-cadherin-IRES-Cre, pAd-CMV-tdTomato-miR-N-cadherin, and pAd-CMV-tdTomato-miR-lacZ. These vectors were transfected into HEK293A cells and adenoviral particles were produced according to the manufacturer's instructions (Invitrogen). Adenovirus particles were concentrated by cesium chloride density gradient centrifugation in an ultracentrifuge (himac CP100WX, Hitachi) at 25,000 g for 2 hours at 4°C, followed by 30,000 g for 3 hours at 4°C. Ad-CMV-Cre (Vector BioLabs) was used as a control for Ad-CMV-DN-N-cadherin-IRES-Cre.

[0050] For N-cadherin knockdown experiments using electroporation, DNA cassettes (tdTomato-miR-N-cadherin and tdTomato-miR-lacZ) were cloned into a modified pCAGGS vector using the Gateway system (Invitrogen). For other knockdown experiments (FAK-KD and L1-CAM-KD), the target sequences of the mouse FAK or L1-CAM gene were inserted into a modified Block-iT Pol II miR RNAi expression vector. The DNA cassettes were cloned into a modified pCAGGS vector using the Gateway system (Invitrogen). All plasmids were prepared using the PureLink HiPure Plasmid Maxiprep Kit (Invitrogen), and their sequences were confirmed by DNA sequencing.

[0051] <Injection of adenovirus vector> Because radial glial cells reside on the ventricular surface and extend long radial processes toward the pial surface, retrograde infection via these processes was induced by injection of small amounts of Ad-Cre into the cortical surface of reporter mice. Thus, Cre-loxP-mediated recombination results in specific and continuous labeling of radial glial cells in the neonatal brain (Merkle et al., 2007, Science 317, 381-384). Radial glial cells were labeled using P0 R26-tdTomato;Dcx-EGFP, Dcx-EGFP, or R26-tdTomato mice using a previously published method with some modifications (Merkle et al., 2007, Science 317, 381-384). Briefly, P0 mice were anesthetized by hypothermia (5 min) or spontaneous inhalation of isoflurane and placed on the platform of a stereotaxic apparatus (David Kopf Instruments) using a skull support. After a scalp incision to expose the parietal bone, 20 nL of adenovirus suspension was injected directly above the surface of the cerebral cortex using the following stereotaxic coordinates: +0.5 mm anterior, +1.0 mm lateral from bregma, and +0.3 mm deep from the skull surface. A beveled, pulled glass micropipette (Wire Troll 5 μl, Drummond Scientific Company) was used for injection. After injection, the scalp was immediately sutured, the mice were returned to their mothers, and monitored until nursing resumed. To examine neuronal maturation, 60 nL of adenovirus suspension was injected into P0 mice as described above using the following stereotaxic coordinates: +0.8 mm anterior, +0.5 mm, +0.2 mm lateral, +1.0 mm from bregma, and +0.3 mm deep from the skull surface. To label V-SVZ cells, 1 μL of adenovirus suspension (Ad-CMV-Cre) was injected into the lateral ventricle of P0 NSE-DTA or C57BL6 / J mice as described above using the following stereotaxic coordinates: +1.8 mm anterior, +1.1 mm lateral from the lambdoid suture, and +2.0 mm deep from the skull surface. Labeling efficiency was as follows:Control (Ad-CMV-Cre) showed 97.7 ± 0.5% of nestin-positive processes at P2 (n = 3 mice); DN-N-cadherin showed 98.0 ± 0.6% at P2 (n = 3 mice); p > 0.05, unpaired t test; control (Ad-CMV-Cre) showed 99.2 ± 0.2% at P9 (n = 4 mice); DN-N-cadherin showed 99.0 ± 0.2% at P9 (n = 3 mice); p > 0.05, unpaired t test; control (Ad-tdTomato-miR-lacZ) showed 98.7 ± 0.4% of nestin-positive processes at P9 (n = 4 mice); N-cadherin-KD showed 97.9 ± 0.4% at P9 (n = 4 mice); p > 0.05, unpaired t test.

[0052] <Postnatal electroporation> V-SVZ cells from P0 ICR, C57BL6 / J, R26-tdTomato, and NSE-DTA mice were labeled using a previously published method (Ota et al., 2014, Nat. Commun. 5, 4532) with some modifications. Briefly, mice were anesthetized by hypothermia (5 min) or spontaneous inhalation of isoflurane and secured to the platform of a stereotaxic injection apparatus (David Kopf Instruments) using a skull support. A solution containing EmGFP-expressing pCAGGS plasmid (7.5 μg / μL per pup) and 0.01% fast green was injected into the lateral ventricle of the right hemisphere (1.8 mm anterior, 1.25 mm lateral, and 2.0 mm deep from the lambdoid suture) and delivered to V-SVZ cells by four 70 V, 50 msec pulses using an electroporator (CUY-21SC, Nepa Gene) and a forceps-type electrode (CUY650P7). The V-SVZ-labeled pups were randomly subjected to cryo-cortical injury and sponge implantation. When mice underwent both adenovirus injection and electroporation on the same day (P0), adenovirus was injected first, followed by electroporation at least 8 h later. The labeling efficiency of V-SVZ cells by pCAGGS-EmGFP electroporation was not statistically different between the test groups at P2 (control, 6.3 ± 1.2% of V-SVZ cells, n = 3 mice; lesioned, 6.3 ± 1.7% of V-SVZ cells, n = 3 mice; p > 0.05, unpaired t test) or at P30 (Ad-Cre; control, 2.7 ± 0.1% of V-SVZ cells, n = 3 mice; Ad-Cre; NSE-DTA, 2.5 ± 0.0% of V-SVZ cells, n = 3 mice; p > 0.05, unpaired t test). The labeling efficiency of DCX-positive cells by pCAGGS-EmGFP electroporation (GFP·DCX double-positive / DCX-positive cells) was 4.0 ± 0.7% of DCX-positive cells (n = 5 mice) in the lesioned cortex at P9.For knockdown studies (N-cadherin knockdown, FAK knockdown, and L1-CAM knockdown), a plasmid solution (7.5 μg / μL per pup) containing 0.01% fast green was injected into the lateral ventricle of the right hemisphere (1.8 mm anterior, 1.25 mm lateral, and 2.0 mm deep from the lambdoid suture), and four 70 V, 50 msec, dorsoventral pulses were delivered using an electroporator (CUY-21SC) and a forceps-type electrode (CUY650P7).

[0053] <Immunoblotting> Immunoblot analysis was performed as previously described (Ota et al., 2014, Nat. Commun. 5, 4532). To confirm the knockdown efficiency of miRNAs (N-cadherin, FAK, and L1-CAM), plasmids expressing cDNAs (N-cadherin, FAK, and L1-CAM) and miRNAs were co-transfected into HEK293T cells using polyethyleneimine. pCAG-MCS2-HA-N-cadherin (Nuriya and Huganir, 2006, J. Neurochem. 97, 652-661) was used as provided. pEGFPC1-mouse FAK (Itoh et al., 2010, Cytoskeleton (Hoboken) 67, 297-308) was used as provided. pCMV6-mouse L1-CAM was purchased from OriGene Technologies. Forty-eight hours after transfection, cells were lysed in lysis buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% NP-40, 0.01% SDS, 10 μg / mL leupeptin). To confirm the expression of neuregulin-1α / 1β / 2, cortical tissue was dissected from wild-type ICR P6 (4 days after injury) mice and homogenized in lysis buffer. The lysate was briefly sonicated and cleared by centrifugation. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) containing 0.01% Tween-20, incubated with primary antibodies overnight at 4°C, and then incubated with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Inc.) for 1 hour at room temperature. Signals were detected and measured using a cooled CCD camera (LAS3000mini, Fujifilm) with enhanced luminal chemiluminescence Western blotting reagent (GE Healthcare). The following primary antibodies were used:Rat anti-HA antibody (1:1,000, Roche), mouse anti-L1-CAM antibody (1:1,000, Abcam), rat anti-GFP antibody (1:1,000, Nacalai Tesque), rabbit anti-neuregulin-1α / 1β / 2 antibody (1:1,000, Santa Cruz Biotechnology), and mouse anti-actin antibody (1:10,000, Millipore). Signal intensity was calculated using ImageJ software.

[0054] <Immunohistological staining> Immunohistochemistry was performed as previously described (Ota et al., 2014, Nat. Commun. 5, 4532). Briefly, brains were fixed by transcardial perfusion with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB) and postfixed overnight at 4°C with the same fixative. Free-floating 60-μm-thick coronal sections were prepared using a vibratome sectioning system (VT1200S, Leica). The sections were incubated in blocking solution (10% normal donkey serum (Millipore) and 0.2% Triton X-100 in phosphate-buffered saline (PBS)) for 40 min at room temperature, with primary antibodies overnight at 4°C, and then with Alexa Fluor-conjugated secondary antibodies (1:500, Invitrogen) for 2 h at room temperature. For the anti-nestin antibody, AffiniPure donkey anti-chicken IgY secondary antibody (Jackson ImmunoResearch Laboratory Inc.) was used. For sponge implantation studies (Figures 3J, 3K, and 4G), 200-μm-thick coronal sections were treated with 100% methanol for 30 min at -30°C, acetone for 30 min at -30°C, 0.3% H2O2 in methanol for 2 h at room temperature, and 50% methanol for 15 min at room temperature before incubation in blocking solution (10% normal donkey serum and 0.5% Triton X-100 in PBS). Signal amplification was performed with a biotin-labeled secondary antibody (Jackson ImmunoResearch Laboratory Inc.) and the Vectastain Elite ABC kit (Vector Laboratories), and signals were visualized using the TSA fluorescence system (PerkinElmer). For Mash1 staining, sections were treated with acetone for 60 s on ice. For double staining of anti-DsRed antibody in combination with anti-Pax6, anti-ErbB4, and anti-Olig2 antibodies, AffiniPure Fab fragment donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.) was used.For double staining of anti-NeuN antibody with anti-parvalbumin (PV), anti-calretinin (CR), and anti-GAD67 antibodies, AffiniPure Fab fragment donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.) was used. The following primary antibodies were used: rabbit anti-Dcx (1:200, Cell Signaling Technology), guinea pig anti-Dcx (1:3,000, Millipore), goat anti-Dcx (1:500, Santa Cruz Biotechnology), rat anti-GFP (1:500, Nacalai), and chicken anti-nestin (1:1,000, Aves). Labs), rabbit anti-DsRed (1:1,000, Clontech), mouse anti-NeuN antibody (1:200, Millipore), mouse anti-CR (1:3,000, Millipore), mouse anti-PV (1:2,000, Sigma), mouse anti-Mash1 (1:100, BD), rabbit anti-Tbr2 (1:200, Abcam), rabbit anti-Pax6 (1:100, Covance), mouse anti-N- The antibodies used were cadherin (1:200, BD), mouse anti-glial fibrillary acidic protein (GFAP) (1:500, Sigma-Aldrich), rabbit anti-Olig2 (1:200, IBL), mouse anti-GAD67 (1:800, Millipore), rabbit anti-ErbB4 (1:300, Abcam), rabbit anti-FAK (1:100, Millipore), and mouse anti-L1-CAM (1:1,000, Abcam). Guinea pig anti-Dlx2 antibody (1:3,000) (Kuwajima et al., 2006, J. Neurosci. 26, 5383–5392) was provided. Hoechst 33342 (1:3,000, Thermo Fisher Scientific) was used for nuclear staining.

[0055] Images of neuronal precursors, radial glial processes, mature neurons, and migratory newborn neurons associated with radial glial processes, sponges, or polyethylene terephthalate (PET) fibers were acquired using an LSM700 confocal laser scanning microscope (Carl Zeiss) with 20x and 40x objectives, scanning at 1 μm intervals. In Figure 3 (A and B), composite images of eight separate fields (two vertical and four horizontal tiles) were acquired with a 20x objective using the tile-scan function of ZEN software (Carl Zeiss). To characterize Dcx-, CR-, PV-, GAD67-, or NeuN-positive neurons, colocalization of signals within the cortex was confirmed by scanning at 1 μm intervals. To measure the amount of EmGFP-positive cells in the V-SVZ and newborn neurons in the lesioned cortex, cells were stereologically counted using a Stereo Investigator system (MBF Bioscience). After adenovirus injection and electroporation, mice underwent randomized cryocortical injury and sponge implantation. To analyze neuronal precursors and migratory newborn neurons, the actual number of cells in every sixth 60-μm-thick coronal section was counted, and then the total number was estimated by multiplying the sum of the counted cells by six. To examine the length and morphology of radial glial processes, three consecutive 60-μm-thick coronal sections were analyzed. To examine the relationship between newborn neurons and radial glial processes, we defined "relationship" as "a distance of less than 2 μm between newborn neurons and radial glial processes," following a previous study (Shikanai et al., 2011, Commun. Integr. Biol. 4, 326-330). For the analysis of mature neurons, we analyzed all EmGFP·NeuN double-positive cells (M2 / M1 / S1HL / S1FL / MPtA / LPtA / S1Tr) in the lesioned sensory and motor cortex (Paxinos et al., 2007, J. Comp. Neurol. 145, 61-83). We counted the actual number of cells in every second 60-μm-thick coronal section and then doubled the sum of the counted cells to estimate the total volume.In the sponge implantation study (Figures 3K and 4G), 200-μm-thick coronal sections were used to preserve the sponge in the lesion area. The morphology of tdTomato-positive radial glial cells was reproduced and quantified using Neurolucida (MBF Bioscience).

[0056] <Transmission electron microscope> Brains from P9 mice infected with control or DN-N-cadherin-expressing adenovirus were fixed by transcardial perfusion with 2.5% glutaraldehyde (GA) and 2% PFA in 0.1 M PB (pH 7.4). The excised brain tissue was cut into 200 μm coronal sections using a vibratome (VT1200S, Leica). The sections were treated with 2% OsO₄ in the same buffer for 2 hours at 4°C. The brain tissue was then dehydrated through graded ethanol concentrations, placed in propylene oxide, and embedded in Durcupan resin for 72 hours at 60°C to ensure polymerization. Serial semithin sections (1.5 μm thick) were cut and stained with 1% toluidine blue. The desired sections were then identified under a light microscope. Ultrathin sections (60-70 nm) were then cut from the semithin sections using a high-resolution microscope (UC6, Leica) and a diamond knife. They were stained with 2% uranyl acetate in distilled water for 15 minutes and modified Sato's lead solution for 5 minutes. The sections were analyzed using a transmission electron microscope (JEM-1400plus, JEOL). AJ-like electron-dense adsorption structures and irregular adhesion lengths were quantified using ImageJ software (National Institutes of Health). Newborn neurons were identified by their dark cytoplasm, numerous free ribosomes, and electron-dense nuclei. Radial glial cells were identified by their sparse nuclei, bright cytoplasm, glycogen granules, and numerous intermediate filaments. The number of cells analyzed was as follows: 21 cells from two mice for the control group and 17 cells from two mice for the DN-N-cadherin group.

[0057] <Time-lapse imaging of damaged brain slices> For time-lapse imaging, an adenoviral vector was injected into P0 R26-tdTomato;Dcx-EGFP mice, and brain sections were prepared 4-5 days after neonatal injury. Briefly, the brain was cut into coronal sections (200 μm thick) using a vibratome (VT1200S, Leica). The sections were placed in a stage-top imaging chamber (Warner Instruments) and were maintained under continuous perfusion with artificial cerebrospinal fluid (aCSF; containing 125 mM NaCl, 26 mM NaHCO3, 3 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 1.25 mM NaH2PO4, and 20 mM glucose; pH 7.4; stored at 38°C; aerated with 95% O2 and 5% CO2) during imaging. Z-stack images (4 z-sections with a 3-5 μm step size) were acquired every 10 minutes for 6-16 hours using a confocal laser microscope (LSM710, Carl Zeiss) equipped with a gallium arsenide phosphide detector. The adhesion time between migrating newborn neurons and radial glial processes was evaluated as the ratio of the process adhesion time during the migration process. To measure the speed, stride length, resting phase, and migration cycle of newborn neurons along radial glial processes in the saved images, unipolar or bipolar newborn neurons in the cortex were traced using ImageJ software (manual tracking plugin). The rate of process extension was analyzed using ImageJ software. All newborn neurons that could be continuously tracked for at least 60 minutes were used for the analysis. For the evaluation of the migration cycle, all newborn neurons that could continuously track at least one cycle of saltatory movement were used. Cells in the "resting" phase were defined as cells whose cell bodies moved slower than 12 μm / h. In Figure 2A, the numbers indicate the time (minutes) from the first frame.

[0058] <Preparation of N-cadherin-Fc sponge> A well-known method (Ajioka et al., 2015, Tissue Eng. Part A 21, 193 - 201) was modified to fabricate gelatin (GE) sponges. 50 μL of 3% GE beMatrix gelatin LS-H (Nitta Gelatin) was added to each well of a 384-well plate and frozen at -20°C. Then, the frozen GE samples were lyophilized at 25°C while centrifuging at 400 rpm (VC-96W, Taitec). After that, the lyophilized GE samples were crosslinked overnight at room temperature with 90% acetone of 25 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Wako). After washing five times with redistilled water, the GE sponges were incubated in Neurobasal medium (GIBCO) for 3 hours. Then, the GE sponges were cut into blocks (1.2×1.2×1.2 mm 3 ), and bound with 10 μg / mL of N-cadherin-Fc (the extracellular domain of mouse N-cadherin is bound to IgG-Fc) or Fc solution (Yue et al., 2010, Biomaterials 31, 5287 - 5296) at 4°C for 24 hours.

[0059] <Fabrication of N-cadherin PET fibers> PET fibers with a diameter of 24 μm (Inoue et al., 2009, J. Biomater. Sci. Polym. Ed. 20, 721 - 736.) were obtained from Toray Industries, Inc. The PET fibers were coated with N-cadherin-Fc (the extracellular domain of mouse N-cadherin is bound to IgG-Fc) or Fc solution (Yue et al., 2010, Biomaterials 31, 5287 - 5296) at 37°C for 1 hour, and then rinsed five times with PBS.

[0060] <Transplantation of N-cadherin sponges or fibers> N-cadherin-Fc sponge or control Fc-sponge, or N-cadherin Fc-PET fiber or control Fc-PET fiber was implanted by a known method (Ajioka et al., 2015, Tissue Eng. Part A 21, 193-201). Briefly, on the 3rd or 10th day after the introduction of freeze injury, mice were anesthetized by natural inhalation of isoflurane. To expose the damaged calvaria, the previous incision was cut and opened with forceps. N-cadherin-Fc or Fc sponge (1.2×1.2×1.2 mm 3 ) was placed into the hole with forceps. For the implantation of PET fibers, fibers with a density of approximately 1.2×1.2×1.2 mm 3 of N-cadherin-Fc or control Fc fibers (length 1.2 mm) were embedded into the hole with forceps. After implantation, the sponge was covered with the calvaria and the scalp was closed. After implantation, the mice were placed on a warm heater for recovery.

[0061] <In vitro cell culture> The stripe assay allowed us to analyze the migratory behavior of single newborn neurons crossing the boundary between a control Fc stripe and an N-cadherin-Fc stripe. For the first stripe, 10 μg / mL N-cadherin-Fc was mixed with 3 μg / mL FITC-conjugated anti-human IgG Fc antibody (Sigma) in Hank's balanced salt solution (HBSS). For the second (control) stripe, 10 μg / mL Fc was mixed with 3 μg / mL anti-human IgG Fc antibody (Sigma) in HBSS. After preincubation of both stripe solutions for 30 min at 4°C with gentle agitation, 100 μL of the first stripe solution was injected into a silicon matrix (50 μm wide) placed in a 35 mm glass-bottom Petri dish. After 30 min of incubation at 37°C, the dish and matrix were rinsed with 500 μL of HBSS, and the matrix was carefully removed. The dish was then coated with 100 μL of the second stripe solution. After 30 min of incubation at 37°C, the dishes were washed three times with HBSS. Neonatal V-SVZ tissue was dissected from WT ICR P0-1 pups and dissociated with trypsin-EDTA (Invitrogen). Cells were washed twice with L-15 medium (GIBCO) containing 40 μg / mL DNase I (Roche) and then transfected with 2 μg of plasmid DNA (pCAGGS-tdTomato-miR-N-cadherin or -LacZ miRNA) using the Amaxa Nucleofector II System (Lonza). The transfected cells were suspended in RPMI-1640 medium (Wako) and allowed to aggregate. The aggregates were then cut into blocks (150–200 μm in diameter), mixed with 50% Matrigel (BD Biosciences) in L-15 medium, and immobilized on stripes.

[0062] For neural culture with N-cadherin-Fc sponge or N-cadherin-Fc fibers, V-SVZ cell aggregates were placed next to N-cadherin-Fc sponge or control Fc sponge, or N-cadherin-Fc fibers or control Fc fibers, in 50% Matrigel. The dishes were stored in a humidified incubator at 37°C and 5% CO2. The aggregate-containing gels were cultured for 48 hours in serum-free Neurobasal medium (GIBCO) containing 2% B-27 supernatant (Invitrogen), 2 mM L-glutamine (GIBCO), and 50 U / mL penicillin-streptomycin (GIBCO).

[0063] Time-lapse video recordings were obtained using an inverted light microscope (Axio-Observer, Carl Zeiss) equipped with a Colibri light-emitting diode light system using a 20x dry objective. Images were automatically acquired every 3 min (Figures 3D and 3E) or 5 min (Figures 2K-2P) for 24 h. Migration speed was quantified using ImageJ software. All newborn neurons that could be continuously tracked for at least 60 min were used in the analysis. For evaluation of the migration cycle, all newborn neurons that could be continuously tracked for at least one cycle of saltatory movement were used. Cells in the "resting" phase were defined as cells whose somata migrated slower than 12 μm / h. In Figures 2K and 3D, the numbers indicate the time (in minutes) from the first frame.

[0064] <Immunocytochemistry> Cultured neurons on coverslips were rinsed with PBS (pH 7.4) and fixed with 4% PFA in 0.1 M PB for 30 min at room temperature. After preincubation for 40 min in blocking solution (10% normal donkey serum (Millipore) and 0.2% Triton X-100 in PBS), cells were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit anti-Dcx (1:200, Cell Signaling Technology), rabbit anti-DsRed (1:1,000, Clontech), and mouse anti-N-cadherin (1:200, BD). Mouse anti-PSA-NCAM antibody (1:1,000) (Seki and Arai, 1991, Neurosci. Res. 12, 503-513) was a kind gift from Dr. Tatsunori Ishi of Tokyo Medical University. Hoechst 33342 (1:3,000, Thermo Fisher Scientific) was used for nuclear staining. Multiply labeled cultured cells were analyzed using an LSM700 confocal laser scanning microscope (Carl Zeiss). For quantification, three or more random fields were selected from each coverslip under a 40x objective. Cell bodies of PSA-NCAM·tdTomato double-positive newborn neurons were tracked, and the expression intensity of N-cadherin was calculated using ZEN software (Carl Zeiss). At least three independent experiments were performed for each quantification.

[0065] <FRETイメージング> FRET imaging of RhoA activity in cultured migrating newborn neurons was performed as previously described (Ota et al., 2014, Nat. Commun. 5, 4532). A FRET probe for RhoA (Raichu-1298X) (Yoshizaki et al., 2003, J. Cell Biol. 162, 223-232) was introduced into cultured V-SVZ-derived newborn neurons by electroporation using the Amaxa Nucleofector II system. Time-lapse imaging of newborn neurons expressing the FRET probe was performed using an LSM700 confocal laser scanning microscope (Carl Zeiss) with a 40x water-immersion objective. FRET ratios (FRET / CFP intensity) were calculated, and final images were generated using the ratio image function in MetaMorph software (Molecular Devices). Baseline RhoA activity was calculated by averaging the basal activity in the leading shaft and defining the average for each cell as 1.0. The degree of RhoA activity in the proximal leading process within the circular region of interest (ROI) (= RhoA prox ) was measured using the Region measurements function of MetaMorph software and normalized to the baseline activity value in each frame (RhoA activity = RhoA prox -1). All bipolar newborn neurons expressing the probe were analyzed in each experiment. Three independent experiments were performed. In Figures 2H and 2I, the numbers indicate the time (minutes) from the first frame.

[0066] <Behavioral testing> Mice underwent quantitative neurobehavioral testing at P30. There were no statistically significant differences in body weight between the test groups. Walking behavior on an elevated hexagonal wire grid was analyzed (foot-fault test). This test assesses motor function related to accurate limb placement and incorporates plantar sensory feedback (Barth et al., 1990, Behav. Brain Res. 39, 73-95). The foot-fault test was performed at 23±1°C. Briefly, mice were placed on an elevated 40-mm-wide hexagonal wire grid and allowed to walk freely. A foot-fault was recorded when one of the four limbs fell into a hole in the grid, causing the mouse to slip or fall. The number of foot-faults for each limb was counted separately for 5 min, and then the ratio of foot-faults by the forelimbs and hindlimbs on the impaired side (left side) to the total number of foot-faults by all four limbs was calculated as a percentage. The test was performed twice and the values ​​were averaged.

[0067] Gait analysis was performed using an automated gait analysis system, Noldus CatWalk XT (Noldus Information Technology), according to the manufacturer's instructions. Briefly, mice were asked to walk on a glass walkway illuminated with a green light source in a dark environment at 23°C ± 1°C. When the sole of the mouse's foot touched the glass surface, the entire surface was reflected internally except for the point of pressure. The contact points of each foot on the glass were illuminated and recorded using a high-speed video camera. The recorded footprints from each test were analyzed using CatWalk XT 10.5 software to generate a series of parameters. For each analysis, at least three successful, continuous walks were recorded for each mouse, and the average was recorded.

[0068] <Experimental Plan> The number of mice, cells, and experimental replicates are described in each figure legend. Except for stereological measurements of the number of EmGFP-positive cells in the V-SVZ and the number of newly generated neurons in the lesioned cortex using the Stereo Investigator system, no special randomization measures were implemented and no blinding was used. No statistical calculations were used to estimate sample size. Experimental sample size was determined according to published studies (Ota et al., 2014, Nat. Commun. 5, 4532; Fujioka et al., 2017, EBioMedicine 16, 195-203). Animals with cryoinjury-induced cortical lesions of 500-600 μm depth were included in the analysis.

[0069] Quantitative and statistical analysis All data are presented ± standard error (± SEM). Two groups were compared using two-tailed paired or independent t-tests, Wilcoxon signed-rank tests, and Mann-Whitney U tests. Comparisons of multiple groups were performed using one-way analysis of variance followed by Tukey's multiple comparison test or Dunnett's test, or Kruskal-Wallis test followed by Steele-Dwas' multiple comparison test or Steele's test. Normality was assessed using the Shapiro-Wilk test. A P value of less than 0.05 was considered statistically significant. The statistical tests and parameters used are as follows: Figure 1G and 1I, (G) n = 3 mice each; unpaired t-test; * p<0.05; (I) n = 4 mice each; unpaired t test. *** p<0.005. Figures 1H and 1J. (H) Control, n=4 mice; DN-N-cad, n=5 mice; paired and independent t-test. *** p<0.005; (J) Control, n=4 mice; N-cad-KD, n=4 mice; paired and independent t-test. * p<0.05, * p<0.01. Figure 1M, Control, n=21 cells; DN-N-cad, n=17 cells; unpaired t-test. ** p<0.01,*** *p<0.005. Figures 2B, 2E, and 2F, Control, n=42 cells from 8 mice; DN-N-cad, n=60 cells from 12 mice; Mann-Whitney U test. *** p<0.005. Figure 2C, Control, n=63 cells from 8 mice; DN-N-cad, n=107 cells from 12 mice; Mann-Whitney U test. *** p<0.005. Figure 2D, Control, n=63 cells from 8 mice; DN-N-cad, n=107 cells from 12 mice; Fisher's exact test. *** p<0.005. Figure 2G, Control, n=39 cells from 8 mice; DN-N-cad, n=37 cells from 10 mice; Mann-Whitney U test. *** p<0.005. Figure 2J, n=9 cells, 3 independent experiments, paired t-test. * p<0.05. Figure 2L, Control, n=15 cells (5 independent experiments); N-cad-KD, n=27 cells (6 independent experiments), paired t-test. *** p<0.005. Figure 2M; Control, n=15 cells (5 independent experiments); N-cad-KD, n=27 cells (6 independent experiments), paired t-test and Wilcoxon signed-rank test. *** p<0.005. Figure 2N, Control, n=16 cells (5 independent experiments); N-cad-KD, n=26 cells (6 independent experiments), Wilcoxon signed-rank test. *** p<0.005. Figure 2O, Control, n=16 cells (5 independent experiments); N-cad-KD, n=23 cells (5 independent experiments), Wilcoxon signed-rank test. *** p<0.005. Figure 2P, Control, n=27 cells (5 independent experiments); N-cad-KD, n=18 cells (4 independent experiments), chi-square test with Yett's continuity correction. * p<0.05. Figure 3C, Control, n = 6 mice; DN-N-cad, n = 7 mice; unpaired t test. *p<0.05. Figure 3E, Control-nonadherent, n=14 cells; Control-adherent, n=19 cells; N-cad-nonadherent, n=19 cells; N-cad-adherent, n=28 cells; three independent experiments; unpaired t-test. *** p<0.005. Figure 3I, P2 (3 dpi), control, n = 7 mice, N-cad, n = 7 mice; P14 (3 dpi), control, n = 6 mice, N-cad, n = 5 mice; 8w (3 dpi), control, n = 7 mice, N-cad, n = 7 mice; P2 (10 dpi), control, n = 4 mice, N-cad, n = 5 mice; unpaired t-test. ** p<0.01, *** p<0.005; control, P2 (3 dpi) vs P14 (3 dpi) or 8w (3 dpi), one-way ANOVA followed by Tukey's multiple comparison test, ###p<0.005; N-cad, (P2 [3 dpi] vs P14 [3 dpi], 8w [3 dpi], or P2 [10 dpi], ##p<0.01, ###p<0.005), (P14 [3 dpi] vs 8w [3 dpi], §§p<0.01); one-way ANOVA followed by Tukey's test. Figure 3K, control, n = 10 mice; N-cad, n = 8 mice; left, unpaired t-test; right, chi-squared test. * p<0.05. Figures 4A-4C, n=10 mice; One-way ANOVA followed by Tukey's test (except for the right side of (A) (Kruskal-Wallis test followed by Steele-Dwas test)). * p<0.05, ** p<0.01, *** p<0.005. Figure 4D. P2 model, control, n = 11 mice; lesion, n = 10 mice; lesion + control-sp, n = 13 mice; lesion + N-cad-sp, n = 14 mice, Kruskal-Wallis test followed by Steele-Dewas test; P14 and 8w models, n = 7 mice each, one-way ANOVA followed by Tukey's test. * p<0.05, *** p<0.005. Figure 4G, Control, n=5; NSE-DTA, n=4; Unpaired t-test. *p<0.05. Figure 4H, Control, n=11 mice; NSE-DTA, n=7 mice; unpaired t test. *** p<0.005. All statistical data, including statistical tests used, standard errors (±SEM) and P values, are presented in the text, figure legends and figures. Error bar values ​​represent ±standard error (±SEM). Litters were randomly assigned to study groups.

[0070] (result) Neonatal radial glial cells maintain their processes after brain injury. We performed cryo-injury on the cerebral cortex on postnatal day 2 (P2) and analyzed the dynamics of radial glial process loss. The density of nestin-positive radial glial processes gradually decreased in the contralateral (non-injured) side, consistent with previous findings (Kriegstein and Alvarez-Buylla, 2009, Annu. Rev. Neurosci. 32, 149-184). In contrast, the density of radial glial processes in the ipsilateral (injured) cortex was highest at 7 days post-injury (dpi) and subsequently decreased, but remained significantly higher than that in the contralateral side at all time points. Furthermore, radial glial processes were longer in the injured brain than in the non-injured brain. These results suggest that neonatal brain injury promotes the persistence of radial glial processes.

[0071] To examine the effects of late-stage injury on radial glial processes, we performed cryocortical lesions in P4, P14, and 8-week-old (8w, adult) mice and analyzed radial glial processes 7 days later. Although nestin-positive processes were maintained in the P4 model, their density was significantly lower than that of the P2 model. No distinct nestin-positive radial glial processes were observed in the P14 and 8w lesion models. These results suggest that radial glial processes only have the potential to be maintained after injury during the neonatal period. Time-lapse imaging of cultured brain slices revealed that retracted radial glial processes could regrow in response to injury. Consistently, these processes in the lesioned brains were significantly longer than those in the unlesioned brains in the P14 and 8w models. Radial glial processes were also observed in neonatal mouse brains after hypoxic and ischemic injury. Taken together, these results indicate that the neonatal brain has the potential to persist radial glial processes after injury.

[0072] Neonatal radial glial cells provide a migratory scaffold for V-SVZ-derived new neurons after brain injury. Freezing injury was induced at P2, and post-injury neurogenesis was investigated at P9 (Figure 1A). Many doublecortin (Dcx)-positive cells with the typical morphology of migratory newborn neurons appeared around the injury site (Figure 1B). These newborn neurons, at least in part derived from the V-SVZ (Figure 1C), were observed to be associated with nestin-positive processes (Figures 1B-1D). To specifically label radial glial processes, adenovirus encoding Cre (Ad-Cre) was injected into the cortical surface of P0 R26-tdTomato;Dcx-EGFP mice (Merkle et al., 2007, Science 317, 381-384) (Figures 1A, 1E). tdTomato fluorescence clearly labeled process-bearing cells expressing the radial glial cell markers Pax6, nestin, and ErbB4 (Schmid et al., 2003, Proc. Natl. Acad. Sci. USA 100, 4251-4256). The cell bodies of these cells, as well as astrocytes and oligodendrocytes, were observed in the V-SVZ and corpus callosum (CC) (Figure 1E'). 55.5% ± 3.1% of Dcx-EGFP-positive newborn neurons migrated radially (toward the lesion), and 96.0% ± 0.3% of these migrating newborn neurons were associated with tdTomato- and nestin-double-positive radial glial processes. Notably, 34.8% ± 4.7% of Dcx-EGFP-positive newborn neurons had their cell bodies aligned with radial glial processes (Figure 1F, 1G). These results suggest that newborn neurons derived from the V-SVZ that migrate radially toward the lesion after neonatal brain injury associate with radial glial processes.

[0073] N-cadherin, a protein involved in regulating cell-cell adhesion, is involved in radial glia-guided migration of newborn neurons in the embryonic cerebral cortex (Kawauchi et al., 2010, Neuron 67, 588-602). We observed that N-cadherin is expressed in both radial glial processes during the neonatal period and in migratory newborn neurons after injury (Figure 1D). To inactivate the function of N-cadherin in radial glia, radial glial processes were infected at post-mortem age (P0) with an adenoviral vector encoding a dominant negative form of N-cadherin (DN-N-cadherin) and Cre (Figures 1A and 1F). DN-N-cadherin expressed in radial glia did not affect radial glial morphology or density at 7 dpi. However, the proportion of newborn neurons associated with radial glial processes expressing DN-N-cadherin was significantly lower in the DN-N-cadherin-infected area compared with the control group (Fig. 1F, 1G). Furthermore, compared with control mice, the density of newborn neurons was significantly reduced in the DN-N-cadherin-infected area and increased in the uninfected area (Fig. 1H). This suggests that newborn neurons prefer radial glial processes lacking DN-N-cadherin for their migration. Specifically downregulating radial glial N-cadherin expression using an adenoviral knockdown (KD) vector also reduced the proportion of newborn neurons associated with radial glial processes and the density of newborn neurons (Fig. 1L and 1J). These results indicate that radial glial N-cadherin is involved in the migration of newborn neurons associated with radial glial processes after injury. Knockdown of FAK and L1-CAM, which are involved in the migration of newborn neurons associated with radial glial processes during embryonic development (Tonosaki et al., 2014, PLoS ONE 9, e86186; Valiente et al., 2011, J. Neurosci. 31, 11678-11691), did not affect the relationship between newborn neurons and radial glial processes.Transmission electron microscopy (TEM) analysis revealed that newborn neurons directly adhered to radial glial processes, and AJ-like electron density structures were observed everywhere (Figure 1K-1K”, red arrows). By DN-N-cadherin expression in radial glia, the density of such structures decreased, and the ratio of irregular adhesions where the cell membranes of newborn neurons and radial glia did not run parallel increased (Figure 1L-1M, blue arrows). These observations suggest that N-cadherin in radial glia is involved in forming proper cell adhesion between radial glial processes and migratory newborn neurons in the neonatal brain. These results indicate that neonatal radial glia are associated with V-SVZ-derived newborn neurons that migrate toward the damaged area after brain injury.

[0074] <N-cadherin promotes RhoA activation and saltatory movement of newborn neurons migrating along radial glial processes> To examine whether newborn neurons use radial glial processes as a scaffold to migrate toward the damaged area, newborn neurons were observed by live imaging at 4-5 dpi in cultured brain slices of R26-tdTomato;Dcx-EGFP mice. Dcx-EGFP-positive newborn neurons extended their leading processes along tdTomato-positive radial glial processes and moved their cell bodies in a saltatory behavior toward the damaged area (Figure 2A and 2A’). Newborn neurons migrating along radial glia expressing DN-N-cadherin had a significantly slower migration speed (Figure 2A and 2B) and frequently detached from radial glial processes (Figure 2A, 2A’ and 2C). Consistent with histological analysis (Figure 1G, 1l), the ratio of newborn neurons not in contact with processes was significantly increased in the DN-N-cadherin group (Figure 2D). These results suggest that N-cadherin in radial glia is involved in the efficient and continuous migration of newborn neurons along radial glial processes toward the damaged area.

[0075] The migration speed of newborn neurons is determined by the cell soma stride length, cell soma stride frequency, and the duration of the resting phase (rest phase) (Ota et al., 2014, Nat. Commun. 5, 4532). Expression of DN-N-cadherin in radial glia significantly reduced cell soma stride length and increased the duration of the resting phase and the migration cycle time of newborn neurons (Figures 2E-2G). These results suggest that neuronal migration along radial glial processes in the injured neonatal brain increases cell soma stride length and the frequency of neuronal saltatory movements via N-cadherin.

[0076] Since RhoA signaling in the swelling region located near the cell body of migratory newborn neurons is known to promote their saltatory motility (Ota et al., 2014, Nat. Commun. 5, 4532), we next observed RhoA activity in the swelling region of migratory newborn neurons coated with N-cadherin-Fc using fluorescence resonance energy transfer (FRET) imaging. RhoA activity in the swelling region of migratory newborn neurons significantly increased upon migration onto scaffolds containing N-cadherin (Figures 2H-2J).

[0077] N-cadherin can interact with various signaling molecules. Next, to examine whether N-cadherin in newborn neurons is also involved in the migration of newborn neurons onto scaffolds containing N-cadherin, an N-cadherin knockdown plasmid was introduced into cultured newborn neurons, and their migratory behavior was analyzed by stripe assay (Figs. 2K-2P). When newborn neurons entered the N-cadherin-Fc stripe, the migration speed of newborn neurons increased significantly (Figs. 2K and 2L). The increased migration speed is thought to be due to both an increase in the stride length of the cell body and a decrease in the time taken for the rest phase in each migration cycle (Figs. 2K-2O). This was consistent with the effect of DN-N-cadherin expression in radial glia (Figs. 2A-2G). When newborn neurons reached the boundary of the N-cadherin-Fc stripe, most of the control cells changed the direction of their leading processes and remained in the region coated with N-cadherin-Fc. The proportion of cells showing this behavior was significantly decreased by N-cadherin knockdown (Figs. 2K and 2P). This suggests that N-cadherin in newborn neurons helps to sustain the directed migration of newborn neurons onto scaffolds containing N-cadherin. These results indicate that N-cadherin promotes RhoA activation and saltatory movement of newborn neurons migrating along radial glial processes.

[0078] <Scaffolds containing N-cadherin promote the recovery of neurological function by increasing the migration and regeneration of V-SVZ-derived neurons after neonatal brain injury> The neonatal V-SVZ supplies newly generated mature neurons to the cerebral cortex under physiological conditions (Le Magueresse et al., 2011, J. Neurosci. 31, 16731-16747) and to the injured striatum and cortex after brain injury (Yang et al., 2007, Ann. Neurol. 61, 199-208; Yang et al., 2008, J. Comp. Neurol. 511, 19-33). Freezing injury to the cerebral cortex increased the number of neural progenitor cells. After injury, the number of newly generated neurons that were Dlx2·Dcx double-positive, but not Tbr2·Dcx double-positive, increased. This suggests that GABAergic newborn neurons are recruited to the injured cortex. Furthermore, neonatal cryoinjury significantly increased the number of EmGFP·NeuN double-positive mature neurons, most of which were GAD67-positive, with fewer Parvalbumin (PV)- or Calretinin-positive neurons. This indicates that these neurons are cortical interneurons derived from the V-SVZ. More than 60% of these neurons were located in cortical layers IV-VI. The number of V-SVZ-derived mature neurons in the cortex was significantly reduced by neonatal radial glia expressing DN-N-cadherin (Figures 3A-3C). This suggests that radial glial processes contribute to the migration and maturation of V-SVZ-derived newborn neurons in the neonatal cortex after injury.

[0079] Next, to test whether an artificial scaffold containing N-cadherin could promote the migration of V-SVZ-derived neurons after brain injury, we developed polyethylene terephthalate (PET) fibers and gelatin sponges conjugated with Fc or N-cadherin-Fc (control or N-cadherin fibers / sponge, respectively). The migration rate of V-SVZ-derived newborn neurons increased when contacted with N-cadherin fibers and sponges in vitro (Figures 3D, 3E, and 3F). We then transplanted N-cadherin fibers or sponges into the injured cortex (Figure 3F). While there was no significant difference in the density of Dcx-positive newborn neurons between control and N-cadherin fibers, the density of newborn neurons within the sponges increased in mice treated with N-cadherin sponges (Figures 3G–3I). This suggests that under the test conditions, N-cadherin sponges support the migration of newborn neurons more efficiently in vivo than N-cadherin fibers.

[0080] To investigate whether N-cadherin sponges promote the migration of newborn neurons in the aged brain, which lacks radial glia, we created cryocortical lesions at P14 or 8w and implanted N-cadherin sponges (Figure 3F). In the control sponge group, the number of newborn neurons reaching the lesion was significantly lower in the P14 and 8w models compared with the P2 model, supporting the idea that radial glial processes are an important scaffold for newborn neurons to migrate toward the lesion (Figures 3H and 3I). Although the absolute number of newborn neurons in the N-cadherin sponge was highest in the P2 model and decreased with age (Figures 3H and 3I), the effect of N-cadherin sponges on promoting the migration of newborn neurons was more pronounced in the aged brain.

[0081] Furthermore, we transplanted N-cadherin sponges into the P2 injury model at 10 dpi and compared the number of newborn neurons within the sponges with that of the 3 dpi transplant group (Fig. 3F). At 4 dpt, the density of newborn neurons in the brain was higher in the P5 transplant group than in the P12 transplant group (Fig. 3I). This suggests that early sponge transplantation has the most beneficial effect on the replenishment of newborn neurons after neonatal brain injury.

[0082] To examine the effect of N-cadherin sponge implantation on neuronal regeneration, V-SVZ cells were labeled by electroporation and analyzed at 28 dpi (Figures 3F, 3J, and 3K). The number of V-SVZ-derived NeuN-positive mature neurons within and around the lesion was significantly higher in mice treated with N-cadherin sponges than in mice treated with control sponges (Figures 3J and 3K). Furthermore, the proportion of V-SVZ-derived NeuN-positive neurons in the upper cortical layer was significantly increased by N-cadherin sponge implantation (Figure 3K). These results suggest that N-cadherin-containing scaffolds promote the regeneration of V-SVZ-derived neurons after neonatal brain injury.

[0083] Finally, we examined the effects of N-cadherin sponge implantation on functional recovery at 28 dpi. We used a catwalk analysis to analyze natural walking behavior. Brain injury caused a decrease in the forelimb contact area ("maximum contact area" and "print area") and an increase in the width between the left and right forelimbs ("base of support") (Figures 4A-4C). Control sponge implantation did not worsen these walking behaviors (Figures 4A-4C), suggesting that sponge implantation had no adverse effects. Notably, N-cadherin sponge implantation improved these impaired walking parameters (Figures 4A-4C). This suggests that N-cadherin sponge promotes functional recovery in addition to neuronal regeneration after neonatal brain injury.

[0084] Next, we performed the foot-fault test (Barth et al., 1990, Behav. Brain Res. 39, 73-95). Freezing cortical injury induced asymmetry in the foot-fault rate at 28 dpi in the P2 injury model. This asymmetry was restored by N-cadherin sponge transplantation but not by control sponge transplantation (Fig. 4D). Furthermore, N-cadherin sponge transplantation significantly improved neurological scores in the P14 model but not in the 8w model (Fig. 4D). Thus, although N-cadherin sponge transplantation can promote the migration of newborn neurons even in the adult brain, the age at which functional recovery is achieved appears to be more limited.

[0085] To further investigate the contribution of endogenous neuronal regeneration from the V-SVZ to functional recovery, we intracerebroventricularly injected Ad-Cre into P0 neuron-specific enolase (NSE)-diphtheria toxin fragment A (DTA) mice (Imayoshi et al., 2008, Nat. Neurosci. 11, 1153-1161; Kobayakawa et al., 2007, Nature 450, 503-508), eliminating their neuronal progeny (Figures 4E-4G). Improvement in stepping rate by N-cadherin sponge implantation was not observed in the Ad-Cre-infected NSE-DTA mice (Figure 4H). These results suggest that the promotion of functional recovery after neonatal brain injury by N-cadherin-containing scaffolds is due to the regeneration of newborn neurons from the V-SVZ.

Claims

1. A method for producing a material for treating brain disorders, comprising a carrier onto which one or more proteins selected from the group consisting of a fusion protein containing all or a partial region of N-cadherin and a fusion protein containing all or a partial region of a protein having homology to N-cadherin are immobilized or coated, the carrier is a gelatin sponge, the fusion protein comprising all or a partial region of N-cadherin and the fusion protein comprising all or a partial region of a protein having homology to N-cadherin are fusion proteins with an Fc region of immunoglobulin, have homophilic binding ability to N-cadherin, and comprise an extracellular domain of N-cadherin or a protein whose amino acid sequence is 90% or more identical to the extracellular domain of N-cadherin; A method for producing a material for treating brain disorders, wherein the immobilization or coating method comprises a step of contacting the carrier with a solution of one or more proteins selected from the group consisting of a fusion protein containing all or a partial region of the N-cadherin and a fusion protein containing all or a partial region of a protein having homology to N-cadherin, at a concentration of 10 to 20 μg / mL.

2. A method for producing a material for regenerating brain neurons, comprising a carrier on which one or more proteins selected from the group consisting of a fusion protein containing all or a partial region of N-cadherin and a fusion protein containing all or a partial region of a protein having homology to N-cadherin are immobilized or coated, the carrier is a gelatin sponge, the fusion protein comprising all or a partial region of N-cadherin and the fusion protein comprising all or a partial region of a protein having homology to N-cadherin are fusion proteins with an Fc region of immunoglobulin, have homophilic binding ability to N-cadherin, and comprise an extracellular domain of N-cadherin or a protein whose amino acid sequence is 90% or more identical to the extracellular domain of N-cadherin; A method for producing a material for regenerating brain neurons, wherein the fixing or coating method comprises a step of contacting the carrier with a solution of one or more proteins selected from the group consisting of a fusion protein containing all or a partial region of the N-cadherin and a fusion protein containing all or a partial region of a protein having homology to N-cadherin, at a concentration of 10 to 20 μg / mL.

3. A method for treating brain disorders (excluding methods for treating human brain disorders), which comprises transplanting into the brain a material for treating brain disorders produced by the method of claim 1.

4. A method for treating brain disorders according to claim 3 (excluding methods for treating human brain disorders), characterized in that neural cells derived from pluripotent stem cells are transplanted into the brain simultaneously with the therapeutic material or after the therapeutic material has been transplanted into the brain.

5. A method for regenerating brain nerve cells (excluding methods for regenerating human brain nerve cells), which comprises transplanting into the brain a material for regenerating brain nerve cells produced by the method of claim 2.

6. A method for regenerating brain neurons according to claim 5, characterized in that neural cells derived from pluripotent stem cells are transplanted into the brain simultaneously with the regenerative material or after the regenerative material has been transplanted into the brain (however, this does not include methods for regenerating human brain neurons).

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