JPWO2008093827A1 - Central nervous system disorder therapeutic agent and method for treating central nervous system disorder - Google Patents

Abstract

EphA receptor ligands such as ephrin-A multimers, for example, ephrin-A1 multimers that multimerize ephrin-A1-Fc fused to IgG (Fc) fragment at the C-terminus of the extracellular domain of ephrin-A1 The ephrin-A1 multimer multimerized using the Coiled-Coil domain site (hTSP5cc) derived from the body and human thrombospondin 5 is injected into the lateral ventricle or striatum to differentiate neural stem cells in the central nervous system. It proliferates and migrates, and recovers damage caused by central nervous degeneration such as Parkinson's disease and striatal nigra degeneration. [Selection] Figure 14

Description

  The present invention relates to a therapeutic agent for central nervous system disorder and a method for treating central nervous system disorder.

  Parkinson's disease is increasing with the aging of the nation, and the prevalence is estimated to be about 100 per 100,000 population, and there are currently about 120,000 patients in Japan. The principle of current treatment is to supplement nigrostriatal dopamine, which is under-produced due to degeneration of dopamine-producing cells, and to correct acetylcholine, which is relatively dominant in the striatum. . For this reason, levodopa preparations and anticholinergic drugs are widely used as first-line drugs and often have dramatic effects. However, in Parkinson's disease, which is a neurodegenerative disease, dopaminergic cells are eventually killed. Therefore, the above treatment is symptomatic and not a fundamental treatment.

  Attempts to transplant fetal substantia nigra cells for the fundamental treatment of Parkinson's disease, but standard treatment due to ethical issues or the availability of sufficient transplanted tissue to meet the number of patients It is not considered. In transplanted clinical trials of cultured fetal substantia nigra cells or fetal substantia nigra tissue conducted in the United States (Non-Patent Documents 1 and 2), there was a slight improvement in patients under 60 years of age or patients with mild symptoms. It was only.

  Furthermore, differentiation induction of human ES cells into dopaminergic neurons and transplantation of the differentiated cells are considered. In studies on monkeys, it is possible to obtain a sufficient amount of dopaminergic cells and symptom improvement has been observed (Non-patent Document 3), but taking into consideration cell differentiation control and tumorigenesis of transplanted cells. Therefore, it must be carefully applied to humans. In addition, this method is a transplantation of cells derived from another person, and causes immunological rejection and ethical problems as in the case of transplantation of fetal substantia nigra cells.

  As a third method, there is an attempt to regenerate a damaged or degenerated central nerve by stimulating neural stem cells (NSC) existing in the subventricular layer and hippocampus in an adult brain. This method mainly includes two types of methods. One is a method in which neural stem cells are cultured, proliferated and differentiated, and then returned to the striatum, and the other induces migration into the striatum without taking the neural stem cells out of the body to become dopaminergic cells. Differentiate. In the former, it is impossible to obtain a sufficient number of cells, and there is no prospect of clinical application. With respect to the latter, various bioactive factors have so far been used with limited success. TGFα, EGF, and FGF2 cause proliferation and migration of neural stem cells and differentiation into glial cells, but do not increase dopaminergic nerves in the striatum (Non-patent Documents 4, 5, and 6). GDNF is a factor with neuroprotective and nerve regeneration effects. Intraventricular injection is ineffective in small-scale clinical trials, but intrastriatal injection is considered effective. Recently, a clinical trial was conducted at Double Blind, but there was a difference between the drug group and the control group. There was not (nonpatent literature 7). Neuroimmunophilin is said to have a nerve regeneration effect, but the mechanism of action is unclear, and it is said that the effect is not clear in short-term tests in monkeys and humans (Non-patent Documents 8 and 9).

  Under such circumstances, the present inventors proliferate the subventricular neural stem cells localized in the lateral ventricular side by injecting into the ventricle a factor that causes proliferation, migration, and differentiation of neural stem cells. Based on the assumption that it can be differentiated into dopaminergic neurons after migrating to the striatum, it has been earnestly repeated.

  Accordingly, attention has been focused on the FGF receptor, which is a receptor for the above-mentioned FGF2, which is said to be involved in the proliferation of various somatic stem cells including neural stem cells, and activated EphA4 and FGF receptor. It was found that the body forms a complex and is more active with each other (Non-patent Document 10). However, the signal molecules downstream of this complex include MAPK, PI-3 kinase, and Rho-GEF, and what effect the control of these signal molecules has on the proliferation, migration and differentiation of somatic stem cells. It was unknown whether it played.

  It is well known that Eph is a transmembrane tyrosine kinase, as described later, and is activated by the binding of its ligand ephrin (for example, Non-Patent Document 11). So far, the culture solution of EphA ligand ephrin-A1 and ephrin-A2 extracellular domain and IgG (Fc) (IgG Fc fragment) fusion protein multimerized with anti-IgG (Fc) antibody In addition, there was a report that differentiation of embryonic neural progenitor cells into dopaminergic neurons was promoted by culturing globular cell clusters of embryonic forebrain derived neural progenitor cells and embryonic forebrain slices. Patent Document 12), no action involved in the proliferation and migration of neural progenitor cells by ephrin-A multimers has been reported. On the other hand, attempts have been made to inject ephrin-A monomer directly into the brain (Patent Document 1) and attempts to inject ephrin-B2-Fc and ephrin-B1-Fc into the ventricle ( Non-patent document 13). In the former approach, ephrin-A2 or ephrin-A5 monomer was administered to suppress EphA7 receptor transmission. It is already known that ephrin monomer suppresses signal transduction via Eph (Non-patent Documents 14 and 15). In Patent Document 1, proliferation of neural stem cells was recognized as a result, but it was not possible to say that neural stem cells were differentiated to increase striatal dopamine-producing cells to restore brain function. The latter also attempts to proliferate neural stem cells by blocking the signal transduction of ephrin-B2-Fc and ephrin-B1-Fc and its receptor, EphB. Furthermore, by injecting drugs directly into the ventricle and striatum and stimulating EphA-mediated signal transduction by binding to EphA receptors, it promotes proliferation, migration and differentiation of neural stem cells There was no report of regenerating neuronal cells that had undergone the treatment, and signal transduction (stimulation) mediated by Eph receptors was unlikely to contribute to the regeneration of neurons in the brain.

  As a result of continuing research, the inventors of the present invention multimerized ephrin-A, which is a ligand of EphA4, and injected it directly into the lateral ventricle or striatum, and as a result, dopaminergic neurons in the striatum were found. Recognizing a significant increase, the present invention has been completed. That is, by injecting multimeric ephrin-A (specifically ephrin-A1) into the lateral ventricle or striatum on the affected side of Parkinson model rats once or continuously, Proliferated, migrated, and increased dopaminergic neurons in the affected striatum. Furthermore, the behavioral analysis of the rat showed an improvement in Parkinson's behavior.

International Publication No. WO03 / 040304 Freed, et al., 2001, N Engl J Med 344: 710-9 Olanow, et al., 2003, Ann Neurol 54: 403-14 Takagi, et al., 2005, J Clin Invest 115: 102-9 Martens, et al., 2002, Eur J Neurosci 16: 1045-57 Wang, et al., 2004, Neurosci Lett 364: 154-8 Kuhn, H.G. et al., 1997, J Neurosci 17 (15): 5820-9 Lang, et al., 2006, Ann Neurol 59: 459-66 Gold and Nutt, 2002, Curr Opin Pharmacol 2: 82-6 Tanaka and Ogawa, 2004, Curr Pharm Des 10: 669-77 Yokote, et al., 2005, Proc Natl Acad Sci USA 102: 18866-71 Wilkinson, 2001, Nat Rev Neurosci 2: 155-164 Aoki, et al., 2004, J Biol Chem 279 (31): 32643-32650 Conover, et al., 2000, Nat Neurosci., 2000, 3: 1091-1098 Davis et al., 1994, Science, 266: 816-819 Vearing, and Lackmann, 2005, Growth Factors, 23: 67-76

  The present invention has been made in view of the above-described background art, and an object of the present invention is to differentiate a patient's own neural stem cells in the center to effectively recover a disorder caused by the degenerated central nerve. .

  In order to solve the above problems, in the present invention, a ligand that binds to EphA and stimulates signal transduction from EphA, a specific example is ephrin-A multimer, more specifically, ephrin-A and IgG ( Fc) ephrin-A-Fc fusion protein consisting of fragments, ephrin-A multimer or ephrin-A and ephrin-A fusion protein consisting of TSP5 (thrombospondin 5) coiled-coil domain A multimer is to be injected into the subventricular or striatum. That is, the therapeutic agent for central neuropathy of the present invention is, for example, like an ephrin-A multimer obtained by multimerizing ephrin-A-Fc in which an IgG (Fc) fragment is fused to the C terminus of the ephrin-A extracellular domain. It is a drug consisting of a ligand that binds to EphA and stimulates signal transduction from EphA, and is intended to be directly injected into the lateral ventricle or striatum.

  According to the present invention, neural stem cells in the lower ventricular layer can be proliferated, migrated into the brain including the striatum, and differentiated into cranial nerves to recover various central nervous system disorders.

It is a block diagram of vector pMXs-IG. FIG. 3 is a structural diagram of a plasmid pCAGVSVG in which a surface antigen of vesicular stomatitis virus is expressed under a chicken β-actin promoter. It is the photograph which shows the influence with respect to the proliferation of the neural stem cell (spherical lump) derived from a mouse embryo, (a) is only a culture medium, (b) is only IgG (Fc) multimer, (c) is ephrin-A1 abundant Body (ephrin-A1-Fc multimer), (d) administered FGF2, and (e) administered both ephrin-A1 multimer and FGF2. The bar in the photograph is 50 μm. (A)-(e) is a photograph which shows the image which expanded the part of the frame enclosure in FIG. 1, respectively. The bar in the photograph is 50 μm. It is the figure which showed the uptake | capture of BrdU to the neural stem cell derived from a mouse embryo. Various molecules (described below the horizontal axis) are expressed using retroviral vectors, and each cell group expressing these different molecules is sequentially controlled from the left, control buffer, ephrin-A1 multimer, FGF2, ephrin- A1 multimer + FGF2 drug administered. * Significant as p <0.01 (n = 4) in Tukey's multiple group comparison test when compared to cells infected with retroviruses expressing only vector-derived EGFP treated with the same drug (Vector) The difference is shown. It is the figure which showed the self-renewal ability of the neural stem cell derived from a mouse embryo. As in FIG. 5, each cell group expressing various molecules (described below the horizontal axis) is administered with a control buffer, ephrin-A1 multimer, FGF2, ephrin-A1 multimer + FGF2 drug in order from the left. * Significant as p <0.01 (n = 4) in Tukey's multiple group comparison test when compared to cells infected with retroviruses expressing only vector-derived EGFP treated with the same drug (Vector) The difference is shown. FIG. 2 is a photomicrograph of nestin immunostaining showing proliferation of neural stem cells two days after drug injection into a mature mouse lateral ventricle, with the sagittal section showing the lateral ventricular side. (A) Injected only IgG (Fc) multimer as control, (b) Injected ephrin-A1 multimer, (c) Injected FGF2, (d) ephrin-A1 multimer + FGF2 was injected. The bar in the photo is 1 mm. It is an immunofluorescence micrograph of nestin showing neural stem cell proliferation 5 days after drug injection into the mature mouse lateral ventricle. (A) is an injection of ephrin-A1 multimer (0.75 μg), and (b) is an injection of IgG (Fc) multimer alone (0.75 μg) as a control. The bar in the photograph is 40 μm. It is a figure which shows the increase / decrease in the amount of nestin specific for the neural stem cell 2 days after drug injection into the mature mouse side ventricle. (A) is a Western blotting diagram, and (b) is a diagram in which the concentration of the band is measured. * Indicates p <0.02, and ** indicates p <0.01 indicating a significant difference. Figures (a) and (b) in order from left to right: IgG (Fc) multimer injected only (control), ephrin-A1 multimer injected, FGF2 injected, ephrin-A1 multimer + FGF2 The injection is shown. It is a photograph showing the expression of ephrin-A in mature normal rat subventricular cells. The right side of each photograph shows the lateral ventricle, and the left side shows the striatum from the lower ventricular layer. The bar in the photograph is 50 μm. It is a photograph which shows the expression of EphA in a mature normal rat subventricular cell. The right side of each photograph shows the lateral ventricle, and the left side shows the striatum from the lower ventricular layer. The bar in the photograph is 50 μm. It is a photograph which shows a Tyrosine hydroxylase (TH) positive cell in the striatum 6 weeks after inject | pouring IgG (Fc) multimer as an ephrin-A1 multimer or control into the ventricular side ventricle of Parkinson model rat. (A) and (b) are administered with ephrin-A1 multimer, (a) is the damaged side, (b) is the healthy side of the same rat, and (c) and (d) are IgG. (Fc) A multimer was administered, (c) shows the damaged side and (d) shows the healthy side of the same rat. The bar in the photo is 1 mm. It is a photograph which shows the expression of TH positive cell in a striatum 6 weeks after inject | pouring an ephrin-A1 multimer into the ventricular side ventricle of a Parkinson model rat. (A) shows the impaired side, (b) shows the healthy side of the same rat. The bar in the photograph is 100 μm. The arrow in the photo indicates the location of drug injection. It is a photograph which shows the expression of TH positive cells in a striatum 6 weeks after inject | pouring ephrin-A1 multimer or IgG (Fc) as a control into the impaired side ventricle of a Parkinson model rat. (A) shows the damaged side of a rat injected with IgG (Fc) multimer as a control, and (b) shows the damaged side of a rat injected with ephrin-A1 multimer. The bar in the photograph is 100 μm. The arrow in the photo indicates the location of drug injection. It is a photograph of an indirect double immunofluorescence staining sample for TH and DAT (dopamine transporter) showing the striatum 6 weeks after injecting ephrin-A1 multimer into the impaired side ventricle of Parkinson model rat. (A) is a composite photograph of FIGS. (B) and (c), (b) is a photograph showing TH positive cells, and (c) is a photograph showing DAT positive cells. The bar in the photograph is 10 μm. Distribution of TH-positive cells when ephrin-A1 multimer or IgG (Fc) multimer as a control is continuously infused into the ventricular ventricle of Parkinson model rats for 1 week and intraperitoneal injection of BrdU (Bromodeoxyuridine) at the same time It is a photograph of an indirect double immunofluorescent staining specimen showing the distribution of cells that have incorporated BrdU into the nucleus. Degraded into nuclei incorporating TH positive cytoplasm and BrdU. (A) shows the damaged striatum of the rat injected with ephrin-A1 multimer, and (b) shows the healthy striatum of the rat injected with ephrin-A1 multimer. (C) shows the damaged striatum of the rat injected with IgG (Fc) multimer as a control, and (d) shows the healthy striatum of the rat injected with IgG (Fc) as a control. In (a) and (c), the left picture shows TH-positive cells, the right picture shows the nucleus that incorporated BrdU, and in (b) and (d) the left picture shows the nucleus that incorporated BrdU, and the right picture shows TH positive cells are shown. The bar in the photograph is 100 μm. The relationship between TH positive cells and BrdU positive cells was examined in detail by taking continuous section images under a confocal microscope at a high magnification in the same tissue as in FIG. It is the photograph under the confocal microscope of the indirect triple (TH, BrdU, DAPI) immunofluorescence dyeing | staining sample in the damage side striatum after inject | pouring an ephrin-A1 multimer into a ventricle. (A) is a composite photograph of (b), (c) and (d), (b) is a photograph showing a TH positive cytoplasm, (c) is a photograph showing a nucleus incorporating BrdU, and (d) is a DAPI stain. It is a photograph shown. The arrows shown in the figure indicate cells that have been confirmed to be TH-positive, that is, nerve cells that are dopaminergic cells. The ephrin-A1 multimer or the IgG (Fc) multimer as a control is continuously infused into the impaired ventricle of Parkinson model rats for 1 week, and at the same time, intraperitoneal injection of BrdU is performed, and the striatum 6 weeks after the end of the operation It is a photograph which shows the result of the indirect double immunofluorescence staining by the antibody with respect to NeuN and the antibody with respect to BrdU in a body. (A) is a composite photograph of FIGS. (B) and (c), (b) is a photograph showing a nucleus incorporating BrdU, and (c) is a photograph showing NeuN staining. It is a photograph which shows the tissue staining of the striatum 6 weeks after inject | pouring ephrin-A1 multimer into the ventricular side ventricle of Parkinson model rat. (B) is a photograph showing TH positive cells, (c) is a photograph showing GFAP positive cells, and (d) is stained with DAPI. It is a photograph showing a nucleus. The arrow in FIG. (A) indicates a portion where a TH positive neuron body is confirmed. The bar in the photograph is 50 μm. It is a photograph which shows the result of having investigated the expression of EGFP in a striatum six weeks after inject | pouring an ephrin-A1 multimer and an EGFP expression retrovirus into the impaired side ventricle of Parkinson model rat. (A) shows TH-positive cells, (b) shows EGFP-expressing cells, and (c) shows a synthetic photograph thereof. The circle in the figure shows the site where EGFP-positive and TH-positive nerve cells (nerve fibers) were observed. The bar in the photograph is 50 μm. Injected ephrin-A1 multimer or IgG (Fc) multimer as a control for 1 week into the ventricular ventricle of Parkinson model rats, and simultaneously injected intraperitoneally with BrdU 6 weeks after the end of the operation It is a photograph of the indirect double immunofluorescence dyeing specimen which shows the uptake of BrdU and the distribution of S100β and GFAP in the lower layer. (A) to (d) are photographs showing the damaged side of control rats injected with IgG (Fc) multimers, and (e) to (h) are photographs showing the damaged side of rats injected with ephrin-A1 multimers. (A) and (e) are composite photographs of (b), (c), (d), and (f), (g), and (h), respectively, and (b) and (f) are photographs that show S100β-positive cells, (c (G) is a photograph showing GFAP positive cells, and (d) and (h) are photographs showing BrdU positive cells. The bar in the photograph is 100 μm. FIG. 22 is an enlarged photograph of a part of FIG. 21 (injured side striatum of a rat injected with ephrin-A1 multimer). (A) is a composite photograph of (b) and (c), (b) is a photograph showing S100β positive cells, and (c) is a photograph showing GFAP positive cells. The bar in the photograph is 20 μm. Brain quality after continuous infusion of ephrin-A1 multimer ((e) to (h)) or IgG (Fc) multimer ((a) to (d)) as a control into the ventricular ventricle of Parkinson model rats It is a photograph which shows the structure image of a lower layer. (A) and (e) are composite photographs of (b), (c), (d), and (f), (g), and (h), respectively, (b) and (f) are photographs that show CD133-positive cells, and (c) (g ) Is a photograph showing GFAP-positive cells, and (d) and (h) are photographs showing capillary endothelial cells positive for RECA-1 (Rat Endothelial Cell Antigen-1). The cells indicated by arrows in the figure are CD133-positive and GFAP-positive cells, and are considered to be pluripotent cells or neural stem cells destined to differentiate into the nervous system. A photograph showing the histology of the subventricular layer at the pre-slice two weeks after a single dose of ephrin-A1 multimer or IgG (Fc) multimer as a control in the impaired ventricle of Parkinson model rats is there. (A) to (e) are cases where IgG (Fc) is administered and (f) to (j) are cases where an ephrin-A1 multimer is administered, (a) is a composite photograph of (b) to (e), ( f) is a composite photograph of (g) to (j), (b) and (g) are photographs showing GFAP positive cells, (c) and (h) are photographs showing GFAP and RECA-1 positive cells, (d) ( i) is a photograph showing RECA-1 positive cells, and (e) and (j) are photographs showing all nuclei present. The bar in the photograph is 100 μm. FIG. 25 is an enlarged view of a part surrounded by a square in (c) and (h) of FIG. 24. (a) is a composite photograph of (b) (c), (d) is a composite photograph of (e) (f), (b) (e) is a photograph showing GFAP-positive cells, and (c) (f) is Each is a photograph showing RECA-1 positive cells. The bar in the photograph is 50 μm. The ephrin-A1 multimer is continuously infused into the impaired ventricle of Parkinson model rats for 1 week, and at the same time, intraperitoneal infusion of BrdU is performed, and TH and BrdU stained images in the striatal capillaries 6 weeks later It is a photograph which shows. (A) is the synthetic | combination photograph of the figure (b) and (c), (b) is a photograph which shows a TH positive cell, (c) is a photograph which shows the nucleus which took in BrdU. The arrow in the figure (a) indicates BrdU positive cells extending TH positive neurites. The bar in the photograph is 25 μm. BrdU was injected intraperitoneally only 3 times every 6 hours after a single injection of ephrin-A1 multimer into the impaired side ventricle of Parkinson model rats, and BrdU in the striatum at 1, 7, and 14 days later It is a photograph which shows the movement of a positive cell. The striatum has a forehead cross section and is stained with BrdU and TH. The left side of the figure shows the impaired side ventricular lower layer and the striatum when injected into the impaired side ventricle, and the right side of the figure shows the healthy side lower ventricular layer and the striatum as controls. In each of the left and right sides, the upper row shows the front striatum, and the lower row shows the middle striatum. Moreover, the white line in a figure shows the migration front of a BrdU positive cell, respectively. The bar in the photograph is 100 μm. FIG. 27 shows a control, in which a single injection of IgG (Fc) multimer into the impaired side ventricle of Parkinson model rats, and then BrdU was injected intraperitoneally only 3 times every 6 hours. It is a photograph which shows the motion of a BrdU positive cell in a striatum 14 days after. Frontal cross section of the middle striatum, stained with BrdU and TH. The left side in the figure shows the impaired ventricular lower layer and the striatum when injected into the impaired side ventricle, and the right side in the figure shows the healthy side as a control. In addition, each white line in the figure indicates the front line of the run. The bar in the photograph is 100 μm. In the presence and absence of SU5402, an FGF receptor inhibitor, ephrin-A1 multimer or IgG (Fc) multimer as a control is administered continuously to the injured side ventricle (right end of the figure) for one week. It is a photograph which shows the effect with respect to the distribution of BrdU positive cell when performing the intraperitoneal injection of BrdU. BrdU is detected by indirect immunofluorescence staining using a specific antibody. (A) When IgG (Fc) multimer is administered (control), (b) When only ephrin-A1 multimer is administered, (c) When ephrin-A1 multimer is administered together with SU5402 Indicates. The bar in the photograph is 100 μm. It is a figure which shows the expression of the TH positive cell in a striatum 6 weeks after inject | pouring a chemical | medical agent into a damaged side striatum of Parkinson model rat once. (A) shows the damaged striatum of the rat injected with IgG (Fc) multimer as a control, and (b) shows the healthy striatum. (C) shows the damaged striatum of the rat injected with ephrin-A1 multimer, and (d) shows the healthy striatum. The bar in the photograph is 100 μm. The arrow in the photo indicates the location of drug injection. It is the figure which showed the behavioral analysis before and behind inject | pouring an ephrin-A1 multimer (3 micrograms) or IgG (Fc) multimer (1.5 micrograms) as a control into the impaired side ventricle of a Parkinson model rat. (A) shows an ephrin-A1 multimer administration group, (b) shows an IgG (Fc) multimer administration group. (A) (b) For each panel, the left side shows the analysis before injection, and the right side shows the analysis after 6 weeks of injection. It is the figure which showed the behavioral analysis after inject | pouring an ephrin-A1 multimer (3 micrograms) or IgG (Fc) multimer (1.5 micrograms) as a control into the damage side striatal body of a Parkinson model rat. (A) shows an ephrin-A1 multimer administration group, (b) shows an IgG (Fc) multimer administration group. (A) (b) For each panel, the left side shows the analysis before injection, and the right side shows the analysis after 6 weeks of injection. It is the figure which showed the behavioral analysis before and after inject | pouring an ephrin-A1 multimer or IgG (Fc) multimer as a control into the impaired side ventricle of Parkinson model rat continuously for 1 week. Post-injection behavior analysis starts from the end of one week of injection. It is a figure which shows the gene cassette for producing the ephrin-A1 multimer (ephrin-A1-hTSP5cc multimer) which is another Example. The purified ephrin-A1-hTSP5cc multimer was separated by SDS-polyacrylamide gel electrophoresis according to molecular weight, and then the protein was detected by Ruby staining or Western blotting. (A) (b) shows the case where each purified ephrin-A1-hTSP5cc multimer is separated under non-reduction, and (c) shows the case where it is separated under reduction. Moreover, (a) shows the case where Ruby staining is applied thereafter, and (b) and (c) show the case where Western blotting is performed by applying anti-ephrin-A1 antibody, respectively. The numerical value at the left end indicates the molecular weight marker in kDa. Using E6A-expressing L6 cells (L6 / EphA4), EphA4 phosphorylation efficacy of ephrin-A1-Fc multimer and ephrin-A1-hTSP5cc multimer was examined by Western blotting with anti-phosphotyrosine antibody Show. Lanes 1 and 2 are for ephrin-A1-Fc multimer stimulation, lanes 3 and 4 are for ephrin-A1-hTSP5cc multimer stimulation, lane 5 is untreated, M is molecular weight marker in kDa units It shows with. It is a photograph which shows the TH positive cell in a striatum 4 weeks after inject | pouring PBS as an ephrin-A1 multimer (ephrin-A1-hTSP5cc multimer) or a control into the impaired side ventricle of a Parkinson model rat. (A) and (b) are rats injected with ephrin-A1 multimer, (a) is the damaged side, (b) is the healthy side of the same rat, and (c) and (d) are PBS. (C) shows the injured side and (d) shows the healthy side of the same rat. The bar in the photo is 1 mm. It is a figure which shows the gene cassette for producing the ephrin-A1 multimer (ephrin-A1-W-hTSP5cc multimer) which is another Example. The purified ephrin-A1-W-hTSP5cc multimer was separated by SDS-polyacrylamide gel electrophoresis according to molecular weight and then detected by Western blotting using anti-ephrin-A1 antibody. (A) shows the case of separation under non-reduction, and (b) shows the case of separation under reduction. The numerical value at the left end indicates the molecular weight marker in kDa. Results of EphA4 phosphorylation of ephrin-A1-hTSP5cc multimers and ephrin-A1-W-hTSP5cc multimers studied by Western blotting with anti-phosphotyrosine antibodies using EphA4 expressing L6 cells (L6 / EphA4) Indicates. Lanes 1 and 2 show phosphorylation by the ephrin-A1-hTSP5cc multimer, lanes 3 and 4 show phosphorylation by the ephrin-A1-W-hTSP5cc multimer, and lane 5 shows changes in the case of no treatment. The numerical value at the left end indicates the molecular weight marker in kDa.

  The present invention relates to an ephrin-A multimer, specifically, an ephrin-A multimer obtained by multimerization of an ephrin-A extracellular domain and an ephrin-A-Fc fusion protein comprising an IgG (Fc) fragment. It is intended to improve neural disorders of the central system such as Parkinson's disease by differentiating neural stem cells by direct injection into the chamber or striatum.

  Group A ephrin (ephrin-A) is a GPI-anchored protein and a ligand that performs signal transduction by binding to group A Eph (EphA) (Deroanne, et al., J. Cell Sci., 116). : 1367-1376, Carter, et al., 2002, Nat. Cell Biol. 4,: 565-73). Eph constitutes a large family of transmembrane tyrosine kinases and is divided into two subgroups (EphA and EphB). EphA is classified into nine types, EphA1 to EphA8 and EphA10, and EphB is classified into five types, EphB1 to EphB4 and EphB6. EphA binds to its ligand, ephrin-A, with some exceptions, and EphB binds to its ligand, ephrin-B. EphA-ligand ephrin-A has five types of ephrin-A1-A5, and EphB-ligand ephrin-B has three types of ephrin-B1-B3. Of these receptors, EphA4 is said to bind ephrin-B2 and B3 in addition to ephrin-A (Wilkinson, 2001, Nat Neurosci., 12: 155-164; Palmer, et al., 2003). Genes & Dev 17: 1429-1450, Aasheim et al., 2005, Biochim Biophys Acta 1723: 1-7).

  As described above, ephrin-A1 and ephrin-A2 (each of which is multimerized) are known to induce differentiation of embryonic neural stem cells in culture (Non-patent Document 12). Transplantation of cultured cells is not sufficient to recover damaged neurons. In Patent Document 1, an attempt has been made to inject ephrin-A2 or ephrin-A5 monomer directly into the brain in order to inhibit EphA-mediated signal, and to proliferate neural stem cells. Has been successful, but neural stem cells have not been differentiated to produce dopaminergic cells. The present inventors activated EphA-mediated signals by multimerizing ephrin-A1 and directly injecting it into the lateral ventricle or striatum of mature animals, and the proliferation, differentiation and migration of neural stem cells And succeeded in increasing dopaminergic cells in the striatum. The present invention makes it possible to fundamentally recover central nervous system disorders such as Parkinson's disease, which have been limited to target therapies such as drug administration.

  When ephrin-A binds to EphA as a monomer, it works to suppress signal transduction from EphA (EphA activity), and when it binds as a multimer, signal transduction from EphA (EphA activity) Is activated (Non-Patent Documents 14 and 15). In the present invention, it is necessary to activate (stimulate) signal transduction to induce proliferation, differentiation and migration of neural stem cells, and EphA ligand ephrin-A needs to be used in multimerization. It is. For dimerization, dimer is insufficient, and it is necessary to multimerize to at least trimer or more, and it is preferable to use a multimer such as tetramer or pentamer. But the therapeutic agent of this invention should just contain a trimer, a pentamer, or more multimer, and even if a dimer is contained with them, it does not interfere. Further, ephrin-A may be any one of ephrin-A1 to A5, which is an EphA ligand, and two or more of A1 to A5 may be used in combination.

  For the multimerization, for example, a method of polymerizing a recombinant protein in which human IgG (Fc) is fused to the C-terminus of ephrin-A with an anti-human IgG (Fc) antibody is used for multimerization. The ephrin-A multimer thus obtained is used as a therapeutic agent for central nervous system disorder according to the present invention. However, the multimerization method may be performed by other methods. For example, Coiled-Coil domain of Cartilage Oligomeric Matrix Protein (COMP) is a small peptide consisting of 45 amino acids and is known to cause pentamerization. It has been reported that Fas and Angiopoietin fused with this peptide multimerize and become potent inhibitors or activators of FasL and Tie receptors (Cho, et al., 2004, Proc Natl Acad Sci USA 101: 5547-52; Holler, et al., 2000, J Immnunol Methods 237: 159-73; JP 2006-504405 A). Therefore, it is conceivable to increase the amount by using such a peptide. In addition to this, a method using cartilage matrix protein CMP, MAT or the like disclosed in JP-T-2006-504405 is also applicable. Furthermore, a ephrin-A multimer can be obtained using a Coiled-Coil domain site (hTSP5cc) derived from human thrombospondin-5.

  At this time, as ephrin-A multimers, those that were multimerized using the entire ephrin-A extracellular domain, but those that were multimerized using only the receptor binding domain that is the active center of ephrin-A were used. Also good. Further, as long as the object of the present invention is achieved, a portion other than the receptor binding region may be included.

  In the present invention, the ephrin-A multimer is directly injected into the lateral ventricle or striatum from the ventricle. At the time of injection, it is used as a pharmaceutical composition dissolved or suspended in a medium that does not lose pharmacological activity, such as physiological saline or PBS (phosphate buffer), that is, capable of holding multimers. In addition, if necessary, the ligand (ephrin-A multimer) may be combined with a solution for dissolution in order to dissolve it before use. Infusion is performed by a suitable device such as a stereotaxic brain infusion device, but may be either single dose or continuous infusion such as infusion. The injection site is not particularly limited as long as it is a lateral ventricle or striatum, but it is preferably the side ventricle and injected proximal to the lesion site. Moreover, in injection (administration), additives such as analgesics, preservatives, and buffering agents may be used as appropriate, and they can be contained in the pharmaceutical composition in advance. The injection amount is 0.01 μg / kg to 1 mg / kg, preferably 0.1 μg / kg to 0.1 mg / kg as a main component per time, but is appropriately determined depending on symptoms, sex, age, body weight and the like. Moreover, the administration target can be applied not only to adult humans but also to young humans and other animal species.

  The central nerve disorder which is the subject of the present invention means all disorders involving degeneration or injury of the central nerve (for example, cranial nerve, spinal nerve, etc.), and is not limited by the etiology. Specifically, Parkinson's disease, Parkinson's syndrome, multisystem atrophy (including striatal substantia nigra degeneration), Huntington's chorea, progressive supranuclear paralysis, diffuse Lewy body disease, cortical basal ganglia degeneration , Alzheimer's disease, senile dementia, Pick's disease, frontotemporal lobar dementia, familial dementia, motor neuropathy (eg, amyotrophic lateral sclerosis, familial amyotrophic lateral sclerosis, etc.), Not only demyelinating diseases (eg, multiple sclerosis, general sclerosis, acute disseminated encephalomyelitis, acute cerebellar inflammation, transverse myelitis, Guillain-Barre syndrome, etc.), but also cerebrovascular disorders (eg, stroke) , Cerebral infarction (eg, cerebral thrombus, cerebral embolism, etc.), transient ischemic attack, reperfusion injury, cerebral hemorrhage (eg, hypertensive intracerebral hemorrhage, subarachnoid hemorrhage, etc.), blood volume reducing shock, trauma Shock / head injury and / or cerebrospinal trauma (eg, Nervous dysfunction associated with contusion, intrusion, shearing, compression, laceration, trauma during delivery, infant whiplash syndrome, etc., cerebrospinal disease associated with infection (eg meningitis, encephalitis, influenza encephalopathy, Creutzfeldt-Jakob disease) , Dementia due to AIDS encephalopathy, etc.), toxic substances (arsenic, cadmium, organic mercury, sarin, soman, butane, VX gas, etc.), neurological dysfunction due to radiation, etc., mental disorders (eg, neurosis, psychosomatic disorders, anxiety, schizophrenia) Illness, manic depression, etc.), epilepsy, Meige syndrome, various dystonia, Down's syndrome, sleep disorders (eg, hypersomnia, narcolepsy, sleep apnea syndrome, etc.), etc., particularly preferably, eg, Parkinson's disease, Alzheimer's It is applied to neurological dysfunction associated with diseases, amyotrophic lateral sclerosis or cerebral infarction.

  Next, the present invention will be described in more detail based on the following examples. Needless to say, the present invention is not limited to the following examples.

(Preparation of ephrin-A multimer)
Fusion of human IgG (Fc) and ephrin-A1 (ephrin-A1-Fc fusion: receptor binding region of ephrin-A1 extracellular domain (shown by the nucleotide sequence shown as ephrin-A1 in FIGS. 34 and 38) Recombinant protein fused with human IgG (Fc) at the C-terminus of the same region) (Sigma-Aldrich Co.) and rabbit anti-human IgG (Fc) antibody (Jackson ImmunoResearch Lab. ) Was mixed with a phosphate buffer at a mass ratio of 1: 2.4 and incubated at 4 ° C. for 1 hour or longer to obtain an ephrin-A1 multimer (ephrin-A1-Fc multimer). As for IgG (Fc) used as a control, the same molar number of IgG (Fc) and rabbit anti-human IgG (Fc) antibody as in the ephrin-A1-Fc fusion were used in a mass ratio of 1: 4.8 (in the molar ratio, ephrin- IgG (Fc) was used as a multimer after incubation with A1-Fc and the same ratio of anti-human IgG-Fc antibody).

[Construction of retroviral vectors]
The following retroviral vectors were used to examine the function of receptors in neural stem cells and other cells. The retroviral vector construction method is as follows.

  EphA4 (WT: Wild Type), EphA4 (ΔJM, ΔK) (molecules from which EphA4's paracellular and kinase domains have been deleted), EphA4 (DN) (molecules from which the intracellular domain of EphA4 has been completely deleted), FRS2α (WT: Wild Type), FRS2α (DN) (a molecule having only a myristylation domain and a PTB domain of FRS2: indicated by FRS2 (DN) in the figure), a gene encoding vector pMXs- Marker gene intervening the sequence of IRES (internal ribosomal entry site) in IG (donated by Prof. Kitamura, University of Tokyo; Kitamura, et al., 2003, Exp. Hematol. 31: 1007-1014: Fig. 1) In order to bind to EGFP (enhanced green fluorescence protein), it was inserted into the MCS part. This pMXs-IG construct was combined with pCAGVSVG (plasma in which the surface antigen of vesicular stomatitis virus was expressed under the chicken β-actin promoter: see Fig. 2) and 293 / gpIRES cells (Osaka University, donated by Professor Miyazawa) Transfected. 293 / gpIRES cells are HEK293T cells in which pGag-pol-IRES-bsr produced by Professor Kitamura of the University of Tokyo is stably expressed by transfection. The thus obtained retroviruses having altered cell directivity were collected by centrifugation (140,000 g, 90 minutes), and then again adjusted to a medium for introduced cells.

[Effects on mouse embryonic neural stem cells]
(1) Culture in spherical mass Mouse embryonic day 14.5 embryonic striatum was removed and mouse embryonic neural stem cells were cultured as spherical mass (medium: 50% Dulbecco's MEM / 50% Ham's F12 supplemented with 5 mM HEPES buffer, pH7.4, 0.6% glucose, 25μg / ml insulin, 100μg / ml transferin, 20nM progesterone, 60μM putrescine, 30nM sodium selenite, 20ng / ml FGF2, and 20ng / ml EGF), coated with poly-L-lysine The same number of spherical lumps were seeded on the culture dish. The medium at this time lacks FGF2 and EGF in the above-mentioned medium components, and instead the drugs shown in the figure (None (no addition), IgG (Fc), ephrin-A1 (ephrin-A1-Fc multimer)) , FGF2, and ephrin-A1 + FGF2) were used. FGF2 was used as a positive control for the proliferation effect of neural stem cells, and was also cultured in a medium (None) containing no drug for comparison. After culturing for 2 days, each cell was observed with a phase contrast microscope. The micrographs are shown in FIGS. Moreover, the photograph which expanded the part of each frame of FIG. 3 to FIG. 4 (a)-(e) was shown. As can be seen from FIGS. 3 and 4, when the ephrin-A1 multimer was added, the cell growth was significantly higher than that of the control, and the cells were growing to the extent that there was no difference compared to the addition of FGF2. It was. In addition, at least additive (or synergistic) cell proliferation was observed in the ephrin-A1 multimer + FGF2 addition group. We have found that FGF receptor multimerizes with activated Eph (Non-patent Document 10), and its ligand, FGF2, is a good growth factor for somatic stem cells including neural stem cells. in use. For this reason, FGF2 was used in the above experiments and in the following several experiments for the purpose of contrasting with the action of ephrin-A1 multimers. Although unpublished, FGF2 has a slightly stronger effect on proliferation of neural stem cells when compared with ephrin-A1 multimers, but has found that neural stem cells have little differentiation into neurons.

(2) BrdU incorporation into cells Next, the cultured neural stem cells were examined for incorporation of BrdU that is incorporated only into proliferating cells. The result is shown in FIG. After removing the embryonic striatum of mouse embryo 14.5 days and culturing it as a neuronal cell mass for 3-5 days, EGFP alone or retro expressing EGFP and the molecule shown in the figure (detailed above) at the same time Viral vectors were infected at a multiplicity of infection (MOI) of 5 before being tested. Judging from the green fluorescence of EGFP, it was confirmed that these proteins were expressed in more than 95% of cells. Cells expressing these different molecules are treated with the above medium (EGF and FGF2) containing drugs (control buffer, ephrin-A1 multimer, FGF2, and ephrin-A1 multimer + FGF2) from the left in the figure. The culture medium was removed for 3 days and the uptake of BrdU was measured for 8 hours. The measurement was conducted using a commercially available ELISA kit (Roche Diagnostics Co.). Each bar shown in the figure represents an average value + standard deviation (n = 4). Comparison was made between groups using Tukey's multiple group comparison test. * Indicates that there is a significant difference from p <0.01 (n = 4) in this multi-group comparison test when compared to cells infected with a retrovirus expressing only the vector treated with the same drug. Yes. Expression of molecules that inhibit EphA4 signaling (EphA4 (ΔJM, ΔK) and EphA4 (DN)) suppresses proliferation signals from all the above drugs, and expression of FRS2α-mediated signal suppressor molecules (FRS2α (DN)) Was found to have a similar effect. From these, it can be inferred that when ephrin-A1 multimers are used, signals are transmitted via EphA4 and FRS2α, and that FRS2α is a downstream signaling molecule shared by the FGF receptor and EphA4. .

(3) Self-renewal ability of mouse embryo-derived neural stem cells The self-renewal ability of neural stem cells was measured by the following method. The result is shown in FIG. After removing embryos of embryos on day 1 of mouse embryo 14.5 and culturing them as neurospheres for 3 to 5 days, EGFP alone (Vector) or a retroviral vector that simultaneously expresses EGFP and the molecule shown in the figure Were infected at a multiplicity of infection (MOI) of 5. Judging from the green fluorescence indicating the expression of EGFP, it was confirmed that these proteins were expressed in 95% or more of the cells. Next, the cells after infection were seeded in a 96-well culture dish at a density of 500 cells / hole, and the regeneration ability of neural stem cells was examined using EGFP fluorescence as an index. Immediately after seeding in a 96-well culture dish, ephrin-A1 multimer, FGF2 and ephrin-A1 multimer + FGF2 were added to the medium on the third and fifth days. The addition group of control (IgG (Fc)), ephrin-A1 multimer, FGF2, and ephrin-A1 multimer + FGF2 is shown from the left for each cell expressing various molecules in the figure. In each group, the number of neuronal spheres emitting fluorescence under an inverted fluorescence microscope equipped with an optical filter for EGFP in at least 30 wells was counted between the 7th and 9th days. Each bar shown in the figure represents an average value + standard deviation (n = 4). The measured values were compared between groups by Tukey's multiple group comparison test. As a result, self-re-performance was suppressed by the expression of an inhibitory molecule (EphA4 (DN)) via EphA4 and the expression of an inhibitory molecule (FRS2α (DN)) via FRS2α. Based on these findings, ephrin-A1 multimers promote self-renewal of neural stem cells via EphA4, and self-renewal by signals via the FRS2α molecule, which is a shared downstream molecule of EphA4 and FGF receptors. It turns out that there is a high probability of promoting.

[Injection into mature mouse lateral ventricle]
(1) Proliferation test of mature mouse neural stem cells 1
ephrin-A1 multimer (0.75 μg / side ventricle), IgG (Fc) multimer (0.75 μg / side ventricle), FGF2 (20 ng / side ventricle) or ephrin-A1 multimer and FGF2 mixture The mice were injected into the lateral ventricle of 6-week-old normal C57BL / 6J male mice. The injection was carried out under anesthesia at 0.0 mm behind the crossing point (Bregma) of the coronal and sagittal sutures of the skull, 1.1 mm laterally (both sides), and 1.5 mm deep. Two days after the injection, indirect immunostaining was performed with a specific primary antibody against nestin, a marker of neural stem cells.

  For indirect immunization whole-mount staining against nestin, mice were first perfused with ice-cold saline under anesthesia followed by 4% paraformaldehyde. The mouse brain was then removed and fixed with 4% paraformaldehyde. The bilateral ventricles were cut with a sagittal section, and mouse anti-nestin monoclonal antibody (rat-401: Chemicon International) was used as the first antibody, and the method of Doetsch and Alvarez-buylla et al. (1996, Proc. Natl. Acad. Sci USA: 14895-14900), and whole-mount staining was performed using the DAB method. Then, it observed with the stereoscopic microscope. The micrograph is shown in FIG. (A) is an IgG (Fc) multimer, (b) is an ephrin-A1 multimer, (c) is FGF2, and (d) is an ephrin-A1 multimer and FGF2. Nestin-positive cells are present in the central area where the cells are stained black. As for the degree of nestin staining, the ephrin-A1 multimer is somewhat weaker than FGF2 but has a sufficient effect, and ephrin-A1 multimer + FGF2 has at least an additive effect of both. From these experimental results, it was found that the ephrin-A1 multimer is as effective as FGF2 for Nestin-positive cell proliferation, and that the ephrin-A1 multimer and FGF2 have at least an additive effect.

(2) Proliferation test of mature mouse neural stem cells 2
Next, in the same manner as above, ephrin-A1 multimer (0.75 μg / side ventricle) or IgG (Fc) multimer (0.75 μg / side ventricle) was injected into the normal mouse side ventricle, and 5 days later. Indirect immunofluorescence staining was performed with a specific primary antibody against nestin. The micrograph is shown in FIG. Each photo shows the lateral ventricular aspect at the frontal section. Nestin-positive cells are thinned. An increase in nestin was observed by single injection of ephrin-A1 multimers, indicating that neural stem cells were proliferating.

  Furthermore, in order to quantitatively examine the increase in nestin, immunoblotting was performed using the whole forebrain 2 days after injection of various drugs (FIG. 9). 1% TritonX-100, 5 mM EDTA, 50 mM NaCl, 10 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate and protease inhibitors (1 mM phenylmethylsulfonylfluoride, 1 μM aprotinin, 1 μM) A 50 mM HEPES buffer containing 1 leMeptupin and 1 μM pepstatin A) was used. Proteins immunoprecipitated with a specific antibody against nestin (rabbit anti-nestin polyclonal antibody: Santa Cruz, Cat. # Sc-20978) were separated by SDS-PAGE, blotted onto a PVDF membrane, and the first antibody (mouse) Anti-nestin monoclonal antibody: Chemicon International, rat-401). Western blotting was performed using the ECL Western Blotting Detection System (Amersham Biosciences, Code # RPN2108) according to the manufacturer's manual. The results of Western blotting are shown in FIG. 9 (a), and the concentration analysis of each band is shown in FIG. 9 (b). Figures (a) and (b) show the IgG (Fc) multimer injected only from the left (control), the ephrin-A1 multimer injected, the FGF2 injected, and the ephrin-A1 multimer. Shown are those injected with + FGF2. Statistical analysis of the results using Student's t-test revealed that ephrin-A1 multimer and FGF2 injection significantly increased nestin levels compared to controls (IgG (Fc)). (*: P <0.02, **: p <0.001). The injection of ephrin-A1 multimer + FGF2 showed at least an additive (or synergistic) effect of each single injection. This result supports the above histological findings.

[Expression of ephrin and Eph in normal adult rat brain]
In order to investigate the effects of ephrin-A1 multimers in rats that have established themselves as Parkinson model animals, first, whether ephrin-A and EphA are expressed in the brain of normal rats and are in a functional state Was confirmed in the absence of drug stimulation. Using the primary antibodies specific to various ephrin-A (ephrin-A1 to A5) (Santa Cruz, Cat. # Sc-911; sc-912; sc-1012; sc-914; sc-6075), the DAB method ( ENVISION Kit, DAKO) indirectly immunostained cells in the subventricular ventricle of normal adult rats (7-9 week old Sprague Dawley male rats). The photograph is shown in FIG. It was shown that the cells positively expressed in the photograph were positively expressed, and endogenous ephrin-A1 to A5 were expressed in the vicinity of the lower ventricle.

  Subsequently, primary antibodies specific for various EphA (EphA1-8) (Santa Cruz, Cat. # Sc-925; sc-924; sc-920; sc-921; sc-1014; sc-8172; sc-1015 sc-7287), and indirect immunostaining of the lateral ventricular subventricular cells of normal adult rats (same as above) by the same method as indirect immunostaining for ephrin. The photograph is shown in FIG. It is shown in the photograph that the cells that are darkly stained are expression-positive cells, and endogenous EphA1-A8 that can be stimulated by ephrin-A1 multimers is expressed from the upper cells to the lower ventricular cells. Thus, it was confirmed that the ephrin-A group and the EphA group were expressed around the lateral ventricle in normal mature rats without drug stimulation.

  Next, the following experiment was conducted using rats with clear methods for preparing Parkinson model animals and the measurement criteria for the symptoms.

[Preparation method of Parkinson model rat and its confirmation method]
Sprague Dawley male rats aged 7-9 weeks under anesthesia were treated with 6-hydroxydopamine (6-OHDA) at the rear of the bregma at 2.2 mm, the side 1.5 mm (right side only), and at a depth of 8.0 mm. ) And injected into the right substantia nigra-striatum dopaminergic nerve tract. 6-OHDA was dissolved in physiological saline at 6 μg / μl, and a solution containing ascorbic acid at a final concentration of 0.2% was injected at 24 μg (4 μl) to 30 μg (5 μl) / rat. Apomorphine was injected intraperitoneally (0.5 mg / kg) at least 6 weeks after 6-OHDA injection into the brain, and the number of rotations specific to Parkinson model rats was measured three times in 20-minute units. Rats having an average rotation number of 100 times or more / 20 minutes were selected as disease-determined model rats.

[Indirect immunofluorescence staining]
After the rats are reflux fixed with 4% paraformaldehyde, the brain is removed and fixed in the same solution overnight. Incubate overnight in 20% and 30% sucrose solutions, respectively, then freeze and store at -80 ° C. If necessary, a 40 μm-thick section is prepared with a cryostat. Staining is performed in a small section in a floating section. First, it is washed three times with PBS (phosphate buffered saline) and then permeabilized in PBS containing 0.25% Triton X-100 and 1% rabbit serum for 30 minutes. Subsequently, after washing three times for 10 minutes in PBS containing only 1% rabbit serum, a primary antibody with an appropriate dilution determined in advance was added to a solution having the same composition as the washing solution, followed by 36 ° C. at 36 ° C. Incubate with time sections. Next, after washing with PBS for 15 minutes 3 times, a secondary antibody (Alexa Fluor-conjugated antibody; Molecular Probes; diluted 500-fold) was added, incubated at 4 ° C. for 2 hours, and washed with PBS for 10 minutes 3 times. . If necessary, DAPI staining of cell nuclei is performed at a concentration of 300 nM and washed with PBS. Then mount it on a slide glass.

[Histological analysis of ephrin-A1 multimer injection effect in Parkinson model rats]
(1) Neuronal regeneration by single injection of ephrin-A1 into the lateral ventricle of Parkinson model rats (expression of dopaminergic cells throughout the striatum)
3 μg of ephrin-A1 multimer was injected into the impaired side ventricle of the Parkinson model rat prepared above. The injection into rats was performed under anesthesia at 0.9 mm rear of Bregma, 1.35 mm laterally (this time only on the right side), and 5.0 mm deep. Thereafter, the injection into the rat side ventricle was performed under the same conditions. Six weeks after injection, the presence of striatal dopaminergic cells was examined by indirect immunostaining (DAB method) using a specific antibody (Chemicon International, Cat. # AB152) against tyrosine hydrokinase (TH). . The photograph is shown in FIG. In contrast, the healthy side of the same rat is also shown. Each section (thickness 40 μm) is a frontal section and shows the middle part of the striatum. In each figure, the darkly stained part is a part where TH positive cells are present. In addition, IgG (Fc) multimers were injected into Parkinson model rats as a control, and the presence or absence of striatal dopamine-producing cells was similarly examined. The striatum on the damaged side injected with ephrin-A1 multimer into the ventricle (Fig. (a)) clearly shows the striatum compared with the striatum injected with IgG (Fc) multimer (Fig. (c)). It has been proven that dopaminergic cells are increasing in the body.

(Expression of dopaminergic cells at high magnification)
The ephrin-A1 multimer or IgG (Fc) multimer (3 μg / lateral ventricle) was injected into the impaired side ventricle of Parkinson model rats, and the expression of TH positive cells in the striatum was examined. Injection and indirect fluorescent immunostaining were performed in the same manner as described above. FIG. 13 is a frontal section, and the middle part of the striatum was examined with a confocal microscope after TH staining. 13 (a) and 13 (b) show the right hand side and the left hand side in the same section. FIG. 14 shows photographs comparing the expression of TH positive cells between when IgG (Fc) was injected and when ephrin-A1 multimer was injected. The arrow in each figure indicates the injection location. Each part in each figure is a part where TH positive cells are present. On the damaged side injected with ephrin-A1 multimer, there was clearly an increase in TH-positive cells in the striatum, but there was almost no TH cell expression in rats injected with IgG (Fc) multimer alone. .

  In addition, in order to clarify that TH positive cells are truly dopaminergic neurons, TH and dopaminergic neurons were obtained from slices of the damaged striatum injected with ephrin-A1 multimer as described above. Indirect double immunofluorescence staining was performed on DAT (Dopamine transporter), which is another indicator of cells. In double staining, antibodies specific for TH (rabbit anti-tyrosine hydroxylase polyclonal antibody Chemicon International, Cat. # AB152) and antibodies specific for DAT (Chemicon International, Cat. # MAB369), indirect immunostaining was performed using antibodies labeled with different fluorescent substances specific for the different animal immunoglobulins as secondary antibodies, and serial sections were taken at 1 μm intervals with a confocal microscope. Was enforced. The result is shown in FIG. (A) is a composite photograph of FIGS. (B) and (c), (b) is a photograph showing TH positive cells, and (c) is a photograph showing DAT positive cells. It is clearly recognized that TH and DAT are co-localized, and it is clear that TH-positive cells are also positive for DAT. TH is positive in both adrenergic and dopaminergic neurons, whereas DAT is positive only in dopaminergic neurons. In other words, it can be said that the regenerated nerve cells are surely dopaminergic. As shown in the figure, TH-positive cells that appeared in the striatum after administration of ephrin-A multimers are DAT-positive cells, confirming that ephrin-A multimers induce dopaminergic neurons. It was done.

(2) Neuronal regeneration by continuous injection of ephrin-A1 into the lateral ventricle of Parkinson model rats (BrdU uptake cell distribution and differentiation)
ephrin-A1 multimer or IgG (Fc) multimer is infused continuously into the impaired ventricle at 3 μg / day for 1 week, and immediately after the start of infusion, BrdU is intraperitoneally administered at 80 mg / kg / dose for 12 weeks. Infused every hour. The brain was taken out 6 weeks after the start of drug injection, and histological analysis was performed by indirect double immunofluorescence staining. In double staining, an antibody specific for TH (the rabbit anti-tyrosine hydroxylase polyclonal antibody) and an antibody specific for BrdU (mouse anti-BrdU monoclonal antibody DAKO, Code #) produced in two different animals as primary antibodies. m0744), indirect immunostaining was performed using antibodies labeled with different fluorescent substances specific for the different animal immunoglobulins as secondary antibodies, and serial sections were taken at 1 μm intervals with a confocal microscope. . The photograph is shown in FIG.16 and FIG.17. (A) and (b) of FIG. 16 are those in which an ephrin-A1 multimer is injected, (a) shows the impaired side and (b) shows the healthy side. Also, (c) and (d) in the figure are those in which an IgG (Fc) multimer was injected, (c) shows the impaired side and (d) shows the healthy side. (A)-(d) Each figure has decomposed | disassembled and shown to the nucleus into which TH positive cytoplasm and BrdU were taken in. In the same figure (a) and (c), the left picture shows TH-positive cells, the right picture shows BrdU, and in the same figures (b) and (d), the left pictures show BrdU, and the right pictures show TH-positive cells. Show. FIG. 17 shows the co-expression of triple (TH, BrdU, DAPI (4'-6-Diamidino-2-phenylindole)) indirect immunofluorescent staining specimens in the damaged striatum after injection of ephrin-A1 multimers into Parkinson model rats. An enlarged photograph of a part of the focused micrograph (FIG. 16 (a)) is decomposed into TH positive cytoplasm (b), nucleus (c) incorporating BrdU and DAPI (all nuclei) (d). It is the photograph shown. In FIG. 16, the white spots are nuclei that have incorporated BrdU, and the whole is white, indicating TH-expressing cells. Rats injected with ephrin-A1 multimer had more BrdU positive cells in the lower ventricular layer and striatum than IgG (Fc) multimer injected rats, and TH positive cells also appeared in the striatum It can be seen that it has come (see FIG. 16A). Moreover, the arrows shown in FIG. 17 indicate cells that have been confirmed to be TH-positive, that is, nerve cells that are dopaminergic cells. From this figure, it can be seen that about 50% of the cells incorporating BrdU are TH positive, that is, many of the neural stem cells are differentiated into nerve cells. Based on the above results, ephrin-A1 multimer injection stimulates neural stem cells in the lower ventricle to proliferate, migrate into the damaged striatum, and a significant percentage of cells are induced to differentiate into dopaminergic cells. it is conceivable that.

  BrdU is taken up into the nucleus of cells that have undergone cell division during the BrdU injection period (first week). When comparing the impaired side with the healthy side, the number of BrdU positive cells increased slightly on the damaged side even in the control injected with IgG (Fc) multimer, but the BrdU positive cells markedly increased by ephrin-A1 multimer injection. Increased on the disabled side. In addition, BrdU positive cells are markedly increased in the lower ventricle, and are distributed more sparsely in the striatum as it becomes more distal to the lower ventricle. This indicates that there is a high possibility that neural stem cells in the lower ventricle migrate into the striatum.

(Differentiation of neural stem cells into neurons in the striatum)
In order to further confirm the differentiation of neural stem cells into neurons using an index other than TH, the following experiment was performed. Continuous injection of ephrin-A1 multimer or IgG (Fc) multimer at 3 μg / day for 1 week in the impaired ventricle of Parkinson model rats, and BrdU intraperitoneally every 12 hours for 1 week immediately after the start of the injection Were injected at 80 mg / kg / dose. Indirect double immunofluorescence staining was performed on striatal sections 6 weeks after the start of drug injection using an antibody against BrdU and an antibody against NeuN as described above. The result is shown in FIG. Since NeuN is stained with nuclei, it has the advantage of being easily correlated with BrdU. Similar to the above experimental results, about 50% of BrdU positive cells were NeuN positive. BrdU-positive cells were originally neural stem cells, and many were found to differentiate into NeuN-positive neurons.

  Next, the presence of both cells in the striatum was examined using staining of TH which is an indicator of dopaminergic neurons and GFAP (glial fibrillary acidic protein) which is an indicator of neural stem cells and astrocytes. The result is shown in FIG. FIG. 19 is the same as described above using a TH-specific antibody (Chemicon International, Cat. # MAB318) and a GFAP-specific antibody (DAKO, cat. # Z0334) for the striatum section obtained above. After indirect double immunofluorescence staining, the nucleus is a photograph of fluorescence staining using DAPI and examined with a confocal microscope. (A) is a composite photograph of FIGS. (B), (c) and (d), (b) is a photograph showing TH positive cells, (c) is a photograph showing GFAP-stained cells, and FIG. (D) is a photograph showing a nucleus. Mixed with GFAP-positive cells (mixture of both neural stem cells and astrocytes), TH-positive nerve cell fibers and cell bodies of dopaminergic neurons as indicated by arrows were observed.

  Moreover, since retroviruses are taken up only by proliferating cells, it was examined whether retroviruses injected into the lateral ventricle were differentiated into neurons after being taken up by neural stem cells. When the ephrin-A1 multimer was injected into the lateral ventricle, an EGFP-expressing retrovirus was simultaneously injected into the lateral ventricle, and the staining of the striatum with TH and EGFP expression were examined with a confocal microscope. The result is shown in FIG. (A) is a photograph showing TH positive cells, (b) is a photograph showing EGFP-expressing cells, and (c) is a composite photograph thereof. The retroviral vector incorporates an expression-inducing sequence for EGFP (enhanced green fluorescence protein). Retroviruses integrate into the nucleus only in proliferating cells and express EGFP in those cells. EGFP positive and TH positive nerve cells (nerve fibers) were observed in the circled part of the figure. From the above, it was confirmed that at least some neural stem cells were in direct contact with the ventricle, and dopaminergic neurons derived from these neural stem cells appeared in the striatum.

  From the above various experiments, it was confirmed that when ephrin-A1 multimers were injected into the lateral ventricle, dopaminergic neurons appeared in the striatum due to neural stem cell differentiation.

(Proliferation and differentiation of neural stem cells in the lateral ventricular subventricle)
ephrin-A1 multimer or IgG (Fc) multimer as a control is infused continuously into the impaired ventricle at 3 μg / day for 1 week, and immediately after the infusion, BrdU is intraperitoneally administered at 80 mg / kg / dose for 1 week. Were injected every 12 hours. After 6 weeks from the start of drug injection, the brain was taken out, and histological analysis was performed by indirect triple immunofluorescence staining of the rat lateral ventricular lower ventricle. In the staining, as the primary antibody, an antibody specific to S100β (Mouse monoclonal antibody Clone SH-B1, Sigma Cat # 2532), an antibody specific to GFAP (Rabbit polyclonal antibody, DAKO Cat # Z0334), and specific to BrdU Using an antibody (Sheep polyclonal antibody, abcam Cat # ab1893), as a secondary antibody, indirect immunostaining is performed using antibodies that are specific to these different animal immunoglobulins and labeled with different fluorescent substances, and then using a confocal microscope Serial section photography was performed at 1 μm intervals. The results are shown in FIGS. 21 (a) to (d) are photographs showing the damaged side of a control rat injected with IgG (Fc), and FIGS. 21 (e) to (h) are photographs showing the damaged side of a rat injected with ephrin-A1 multimers. (A) and (e) are composite photographs of (b), (c), (d), and (f), (g), and (h), respectively, and (b) and (f) are photographs that show S100β-positive cells, (c (G) is a photograph showing GFAP positive cells, and (d) and (h) are photographs showing BrdU positive cells. FIG. 22 is an enlarged photograph of the damaged striatum body injected with ephrin-A1 multimer, (a) is a composite photograph of FIGS. (B) and (c), and (b) shows S100β positive cells. Photograph (c) is a photograph showing GFAP positive cells.

  As shown in FIGS. 21 (a) to 21 (d), the lower ventricular layer of the control rat is composed of S100β-positive one-layered ependrite cells and cerebral ventricular lower layer cells considered to be GFAP-positive neural stem cells. There are few cells that are. However, injection of ephrin-A1 multimer significantly increases S100β-positive and GFAP-positive cells as shown in FIGS. 21 (e) to (h). BrdU, which is taken up only by the nuclei of proliferating cells, is taken up by GFAP-positive cells and should not be taken up by itself (Spassky et al., 2005, J Neurosci 25: 10-18). It is clear that In addition, as shown in FIG. 22, S100β-positive and GFAP-positive cells have appeared in the striatum by injecting ephrin-A1 multimers into the lateral ventricle, but there are also cells that are only GFAP-positive. . In the striatum, S100β is positive in cells that have differentiated into astrocytes (Raponi et al, 2007, Glia 55: 165-177), and cells that are positive only for GFAP still retain the properties of neural stem cells It is considered a thing. From the above, neural stem cell Niche composed of ependymocytes (S100β positive) and subventricular neural stem cells (GFAP positive) was reconstructed by intraventricular injection of ephrin-A1 multimer, It can be inferred that remarkable proliferation of neural stem cells and differentiation into neurons (especially dopaminergic neurons) and astrocytes occur.

  Furthermore, we investigated whether pluripotent stem cells appeared in the subventricular layer by lateral ventricular injection of ephrin-A1 multimers. Antibodies specific for CD133 (Goat polyclonal antibody, Santa Cruz Biotech Cat # sc-23797), antibodies specific for GFAP (Rabbit polyclonal antibody, DAKO Cat # z0334), and antibodies specific for RECA-1 (Mouse monoclonal antibody) , Santa Cruz Biotech Cat # sc-23797) and indirect triple immunofluorescence staining of subventricular tissues using antibodies labeled with different fluorescent substances specific for the different animal immunoglobulins as secondary antibodies The photo was taken with a confocal microscope. The result is shown in FIG. 23 (a) to (d) are photographs showing the damaged side of control rats injected with IgG (Fc), and FIGS. 23 (e) to (h) show the damaged side of rats injected with ephrin-A1 multimers. (A) (e) is a composite photograph of (b) (c) (d) and (f) (g) (h), and (b) (f) is a photograph showing CD133 positive cells, (C) and (g) are photographs showing GFAP positive cells, and (d) and (h) are photographs showing RECA-1 positive vascular endothelial cells. From these photographs, pluripotent stem cells (CD133 positive cells) and so-called neural stem cells (CD133 positive and GFAP positive cells) that were destined to differentiate into the nervous system by intracerebroventricular injection of ephrin-A1 multimers; arrows in the figure ) Increases in the lower ventricle and moves into the striatum. In addition, in the lower ventricular layer on the side where ephrin-A1 multimer was injected, such cells were observed along the vascular endothelial cells, but in the control-injected brain, such cells were not observed along the blood vessels. .

(3) Collaboration between capillary regeneration and nerve cell regeneration by ephrin-A1 injection into the lateral ventricle of Parkinson model rats)
A single injection of 4 μg of ephrin-A1 multimer was made into the ventricular ventricle of Parkinson model rats. Two weeks after injection, the increase in blood vessels in the lower ventricle was examined by indirect double immunofluorescence staining using antibodies specific for rat vascular endothelial cells RECA-1 and GFAP, and nuclei were examined by staining with DAPI. It was. The result is shown in FIG. 25 in which the portion surrounded by the square in FIGS. 24 and 24 (c) (h) is enlarged. By ephrin-A1 injection, blood vessels from the lower ventricle into the striatum increase as shown in FIG. 24 (i), and vascular endothelial cells (RECA) as shown in FIG. 24 (g) (h) and FIG. -1 positive cells) and GFAP positive cells (cells thought to be neural stem cells or astrocytes) were increased. Thus, it was found that the capillaries in the brain and neural stem cells are in a close relationship.

  Next, the relationship between capillaries and neurons was examined in detail. An IgG (Fc) multimer or ephrin-A1 multimer is infused continuously into the impaired ventricle at 3 μg / day for 1 week. Immediately after the start of the infusion, BrdU is intraperitoneally administered at 80 mg / kg / dose for 12 weeks. Infused every hour. Six weeks after the start of drug injection, the antibody specific for TH (the rabbit anti-tyrosine hydroxylase polyclonal antibody) and the antibody specific for BrdU (mouse anti-BrdU monoclonal antibody DAKO, Code # m0744) were used in the same manner as described above. Indirect double immunofluorescence staining of striatum sections was performed. The result is shown in FIG. (B) is a photograph showing TH positive cells, (c) is a photograph showing cells in which BrdU has been incorporated into the nucleus, and (a) is a composite photograph thereof. It can be seen that BrdU is taken up in the nucleus of vascular endothelial cells in a form surrounding capillaries (Cap) in the striatum, and that some BrdU positive cells (white arrows) extend TH positive neurites. From these results, it is considered that under the intraventricular administration of ephrin-A1, there is a high possibility that dopaminergic neurons and capillary endothelial cells are differentiated from the same stem cells. In addition, both of these cells may be derived from CD133 positive cells appearing around the blood vessel shown in FIG.

(4) Neural stem cell migration effect of ephrin-A1 injection into the lateral ventricle of the Parkinson model rat (Migration of mature rat neural stem cells from the subventricular layer to the striatum: pulse-chase experiment)
After injecting 3 (g ephrin-A1 multimer or IgG (Fc) multimer at a time into Parkinson model rat injured side ventricle, inject 3 times every 6 hours into the abdominal cavity. The movement of BrdU positive cells in the striatum was examined after 1 day, 7 days and 14 days, and the results are shown in Fig. 27 and Fig. 28. Fig. 27 shows the case where ephrin-A1 multimer was injected, 27 shows the case where an IgG (Fc) multimer as a control was injected, and each photograph was obtained by staining cells in the lateral ventricle and the striatum by indirect double immunofluorescence staining, as shown in FIG. In both left and right photos, the upper row shows the anterior striatum, the lower row shows its middle striatum, and the migration front of BrdU positive cells in the damaged striatum by injecting ephrin-A1 multimers into the damaged ventricle. (Indicated by the white line) moved from the lower ventricular layer to the inside of the striatum with time (left side of Fig. 27). In the striatum, BrdU-positive cells were sparse, and almost no migration into the striatum was observed (right side of Fig. 27.) When IgG (Fc) multimers were injected into the damaged ventricle Therefore, almost no migration of BrdU-positive cells into the striatum was observed in the damaged striatum.In conclusion, injection of ephrin-A1 into the striatum was caused by intraventricular injection of neural stem cells into the striatum. It was proved that they were involved.

  It has been previously reported by the present inventors that signals of the ephrin-A / Eph system also affect the FGF receptor (Yokote, et al., 2005, Proc Natl Acad Sci USA 102: 18866-71) . Therefore, we investigated whether FGF receptor-mediated signals are involved in neural stem cell migration from the subventricular layer to the striatum caused by ephrin-A1. In the presence or absence of SU5402, an FGF receptor inhibitor, ephrin-A1 multimer or IgG (Fc) multimer is injected into the impaired ventricle (right side of the figure) at a dose of 3 μg / day for 1 week. During the injection, BrdU was injected intraperitoneally, and the effects of ephrin-A1 multimer and SU5402 were examined by the distribution of BrdU positive cells. FIG. 29 shows indirect double immunofluorescence staining of cells in the lateral ventricular sublayer. FIG. 29 (a) shows only the control IgG (Fc) multimer, and FIG. 29 (b) shows the ephrin-A1 abundance. FIG. 3 (c) shows the ephrin-A1 multimer and SU5402 administered, respectively. As shown in the figure (c), in the presence of SU5402, the migration of BrdU positive cells into the striatum is markedly suppressed, and at least part of the neural stem cell migration effect of ephrin-A1 is mediated by the FGF receptor. It is thought that.

(5) Nerve cell regeneration effect of single injection of ephrin-A1 multimer into the striatum of Parkinson model rat Injection of 1.5 μg of IgG (Fc) multimer or ephrin-A1 multimer into the striatum of Parkinson model rat injured side Then, 6 weeks after the injection, the brain was taken out and the expression of dopaminergic nerves was examined histologically with a TH-specific antibody. The result is shown in FIG. FIGS. 30 (a) and 30 (b) show control rats injected with IgG (Fc), and FIGS. 30 (c) and (d) show rats injected with ephrin-A1 multimers. FIGS. Each of the photographs shows the handicapped side and FIGS. (B) and (d) are photographs showing the healthy side. Moreover, the arrow of a figure has shown the chemical | medical agent injection | pouring location. Appearance of dopaminergic nerves was observed only in the part near the ventricle of the damaged striatum injected with ephrin-A1 multimer (Fig. (a)). The healthy side (FIGS. (B) and (d)) is in the same section as the impaired side and is shown as a control. This suggests that the ephrin-A1 multimer that diffused from the damaged striatum to the lower ventricle layer stimulated the neural stem cells present therein to induce differentiation into dopaminergic neurons.

[Analysis of behavior after administration of ephrin-A1 multimer to Parkinson model rats]
(1) Administration to the rat side ventricle 3 μg of ephrin-A1 multimer or 1.5 μg of IgG (Fc) multimer (same molar amount as ephrin-A1 multimer) is injected into the impaired side ventricle of Parkinson model rats. Then, after 6 weeks, the number of rotations was measured again in units of 20 minutes, and the average value was used as the number of rotations after the treatment of each rat. These results are shown in FIG. Student's t-test showed a significant difference (P <0.01) in the number of rotations between the two, and it was confirmed that Parkinson's symptoms were clearly improved by administration of the ephrin-A1 multimer to the lateral ventricle. .

(2) Administration to rat striatum In the same manner as above, the ephrin-A1 multimer or IgG (Fc) multimer is injected into the impaired striatum of Parkinson model rat, and the behavior of the rat before and after administration is determined. Observed. The result is shown in FIG. When Student's t-test was performed, a significant difference (P <0.01) was observed in the number of rotations of both, and it was confirmed that Parkinson's symptoms were clearly improved by administration into the striatum.

(3) Continuous infusion into rat ventricle In the Parkinson model rat prepared by the above method, IgG (Fc) multimer (dose 2.0 μg / day) or ephrin-A1 multimer (administration) (1.0 μg / day and 2.0 μg / day) were continuously infused with an implantable osmotic pump. After 4 weeks, 8 weeks and 12 weeks from the start of injection, the number of rotations was measured again in units of 20 minutes, and the average value was used as the number of rotations after the ephrin-A1 multimer injection treatment of each rat. The results are shown in FIG.

  The number of rotations after 12 weeks increased in the control IgG (Fc) multimer-injected rats from the pre-injection value, but in ephrin-A1 multimer-injected rats all decreased to less than 50% of the previous value. The effect of ephrin-A1 multimer administration was clearly observed.

  From the various experiments described above, ephrin-A1 multimers are administered into the lateral ventricle or / and the striatum to proliferate, migrate, and differentiate subventricular neural stem cells into the striatum, thereby causing dopaminergic nerves. It became clear to increase and reduce Parkinson's symptoms in rats.

[Preparation of ephrin-A multimer (ephrin-A1-hTSP5cc multimer)]
An ephrin-A1 multimer (ephrin-A1-hTSP5cc multimer) was prepared by genetic engineering using a TSP5 (thrombospondin 5) coiled-coil domain that promotes pentamer formation. The coiled-coil domain that multimerizes ephrin-A1 was derived from human thrombospondin 5 (hTSP5). The ephrin-A1-hTSP5cc multimer is a multimerization of a fusion protein (ephrin-A1-hTSP5cc) in which the hTSP5 coiled-coil domain is fused to the C-terminus of the ephrin-A extracellular domain. The fusion protein molecule ephrin-A1-hTSP5cc binds the extracellular domain of ephrin-A1 and the coiled-coil domain of hTSP5 via the three consecutive amino acid sequences of Flag (peptide epitope), and then the coiled-coil domain of hTSP5 It is a protein with 6 histidine residues bound to the C-terminus. The base sequence encoding the fusion protein molecule ephrin-A1-hTSP5cc is shown in SEQ ID NO: 1. This nucleotide sequence was bound to the marker gene EGFP through the IRES (internal ribosomal entry site) sequence in the aforementioned vector pMXs-IG (FIG. 34). This pMXs-IG construct was then transfected into Example 1 together with the aforementioned pCAGVSVG and also into the aforementioned 293 / gpIRES cells in Example 1 (contributed by Prof. Miyazawa, Osaka University). Thus, a retrovirus expressing ephrin-A1-hTSP5cc was prepared, and NS0 / ephrin-A1-hTSP5cc that stably secreted and produced ephrin-A1-hTSP5cc was prepared by infecting NS0 cells with it. Multiple clones of this cell were cloned, and the supernatant of the clonal cell secreting the largest amount of ephrin-A1-hTSP5cc was used as a material for purifying ephrin-A1-hTSP5cc molecules. This protein molecule was separated using two types of affinity columns, ANTI-FLAG M2 Affinity Gel (SIGMA, cat # A2220) and TALON Superflow Metal Affinity Resin (Clontech, cat # 635506). Further purification was performed by fractionating a molecular weight of 100 kDa or more with Ultra-4 Ultracel-100k (Millipore, cat # UFC805008). In this ephrin-A1 multimer, multiple ephrin-A1 is considered to be multimerized by the coiled-coil domain of hTSP5.

  Next, the purified ephrin-A1-hTSP5cc multimer is separated by SDS-polyacrylamide gel electrophoresis under non-reduction, and then the sensitive Ruby staining method (FIG. 35 (a)) for staining all proteins. Detection was performed by Western blotting using an anti-ephrin-A1 antibody (FIG. 35 (b)). Both test methods detected molecular masses of ephrin-A1-hTSP5cc trimer or higher. The purified ephrin-A1-hTSP5cc multimer was separated by SDS-polyacrylamide gel electrophoresis under reduction and then detected by Western blotting using an anti-ephrin-A1 antibody. A 40 kDa molecule was detected (FIG. 35 (c)). From these results, it was confirmed that the protein was efficiently purified and that ephrin-A1-hTSP5cc formed a multimer as expected.

[EphA4 phosphorylation efficacy of ephrin-A multimers (ephrin-A1-Fc multimers and ephrin-A1-hTSP5cc multimers)]
EphA4 phosphorylation efficacy of ephrin-A1-Fc multimers and ephrin-A1-hTSP5cc multimers, which are multimerized ephrin-A1, was examined using L6 cells (L6 / EphA4) expressing EphA4. L6 / EphA4 was incubated at 37 ° C for 1 hour in a culture solution (Dulbecco's Minimum Essential Medium) in which serum was removed at a final concentration of 0.05 and 0.5 µg / ml of each ephrin-A1 multimer, and the cells were lysed (Example) After treatment with the tissue lysis buffer used in 2), immunoprecipitation was performed with anti-EphA4 antibody, the precipitate was separated by SDS-polyacrylamide electrophoresis, and subjected to Western blotting using anti-phosphotyrosine antibody. Detected. The result is shown in FIG. In both cases, a band appeared around 120 kDa, confirming that EphA4 was phosphorylated almost equally. These two types of ephrin-A1 multimers have the same effect in this cell, and the ephrin-A1-TSP5cc multimer multimerized by the hTSP5 coiled-coil domain is similar to the ephrin-A1-Fc multimer. It can be presumed that it exerts the proliferation, differentiation and migratory action of cranial nerve stem cells.

[Single injection of ephrin-A1-hTSP5cc multimer into the ventricle of Parkinson model rats]
A single injection of 3 μg of the purified ephrin-A1-TSP5cc multimer or PBS into the lateral ventricle adjacent to the damaged striatum of Parkinson model rats, followed by DAB using anti-TH antibody 4 weeks later The striatum was stained. The result is shown in FIG. Figures (a) and (b) show the bilateral striatum of the same rat injected with the ephrin-A1-hTSP5cc multimer. Figures (c) and (d) show the bilateral lines of the same rat injected with the control PBS. Shows the body. Moreover, (a) and (c) show a failure side, respectively, and (b) and (d) show a healthy side, respectively. Dopaminergic neurons are darkly stained.

  By injecting the ephrin-A1-hTSP5cc multimer into the ventricle, it can be seen that dopaminergic neurons are clearly increased in the damaged striatum (see (a) in the figure). On the other hand, dopaminergic neurons are not increased at all in the striatum of rats injected with control PBS (see (c) in the figure). Thus, it can be said that the ephrin-A1-hTSP5cc multimer promotes the regeneration and expression of neural stem cells in the same manner as the ephrin-A1-Fc multimer.

[Preparation of ephrin-A multimer (ephrin-A1-W-hTSP5cc multimer)]
Similarly, ephrin-A1 multimer (ephrin-A1-W-hTSP5cc multimer) in which ephrin-A is highly multimerized using two TSP5 coiled-coil domains that promote pentamer formation in one molecule. ) Was produced by genetic engineering. The ephrin-A1-W-hTSP5cc multimer is a multimer of ephrin-A fusion protein (ephrin-A1-W-hTSP5cc) in which two TSP5 coiled-coil domains are fused to the C-terminal side of the ephrin-A extracellular domain It has become. This fusion protein ephrin-A1-W-hTSP5cc binds the extracellular domain of ephrin-A1 and the hTSP5 coiled-coil domain via a three-continuous amino acid sequence of Flag (peptide epitope), and this hTSP5 coiled-coil domain A protein in which six histidine residues are bound after the second hTSP5 coiled-coil domain is bound to the C-terminal of the second hTSP5 coiled-coil domain via the three consecutive amino acid sequences of the second Flag (peptide epitope). The base sequence encoding the fusion protein ephrin-A1-W-hTSP5cc is shown in SEQ ID NO: 2. This base sequence was bound to the marker gene EGFP via the IRES (internal ribosomal entry site) sequence in the aforementioned vector pMXs-IG (see FIG. 38). Using the thus constructed pMXs-IG construct, a purified protein molecule was obtained in the same manner as in Example 4. The base sequence encoding the fusion protein ephrin-A1-W-hTSP5cc incorporates the base sequence encoding the hTSP5 coiled-coil domain at two positions, and more ephrin-A1 than the multimer of Example 4 The receptor binding region of the protein is multimerized.

  Next, purified ephrin-A1-W-TSP5cc multimers were separated by non-reducing and reducing SDS-polyacrylamide gel electrophoresis, and then detected by Western blotting using anti-ephrin-A1 antibody . The result is shown in FIG. Under non-reduction, a multimer having a molecular weight of about 100 kDa or more was detected (see (a) of the figure), and under reduction, a monomer of about 50 kDa and a small amount of a molecule of about 40 kDa were detected ((b) of the figure). ). ephrin-A1-W-hTSP5cc is a molecule in which two coiled-coil domains of hTSP5 are fused to the C-terminus of ephrin-A1, resulting in a molecule fused with one coiled-coil domain of hTSP5 (ephrin-A1-hTSP5cc It was confirmed that an advanced multimer of ephrin-A1-Fc fusion protein was formed. A molecule of about 40 kDa is considered to be a protein that has been degraded by peptidase or the like.

[EphA4 phosphorylation efficacy of ephrin-A multimers (ephrin-A1-hTSP5cc multimers and ephrin-A1-W-hTSP5cc multimers)]
Using EphA4-expressing L6 cells (L6 / EphA4), the ephrin-A1-hTSP5cc multimer and the ephrin-A1-W-hTSP5cc multimer EphA4 phosphorylation efficacy, It investigated like the said Example 4. The result is shown in FIG. In both cases, a band with almost the same intensity appeared around 120 kDa, confirming that EphA4 was phosphorylated equally. In other words, the ephrin-A1-W-hTSP5cc multimer multimerized by two hTSP5 coiled-coil domains and the ephrin-A1-hTSP5cc multimer multimerized by one hTSP5 coiled-coil domain (Example) It has EphA4 activation potency equivalent to 4) and is considered to exert neural stem cell proliferation, differentiation, and migration.

  In Examples 4 and 5, when an ephrin-A1 multimer is prepared using the hTSP5 coiled-coil domain, a Flag (peptide epitope) is interposed between the receptor binding region of ephrin-A1 and the hTSP5 coiled-coil domain. In the present invention, it is needless to say that other amino acid residues and peptide residues may be interposed as long as the ephrin-A1 multimer can be formed. Similarly, other amino acid residues may be bound to the residues. Moreover, it does not matter if they are not present. The base sequence 1 and the base sequence 2 are intended to include base sequences that have undergone base substitution, base insertion, or base deletion as long as the functions of the present invention are exhibited.

  The present invention provides new therapeutic agents for central neurodegenerative disorders such as Parkinson's disease, Parkinson's syndrome, and Alzheimer's.

Claims (18)

  A therapeutic agent for central neuropathy for ventricular or striatum injection, comprising a ligand that binds to EphA and stimulates signal transduction from EphA.   A therapeutic agent for central nervous system disorder for infusion of ventricles or striatum characterized by comprising Ephrin-A multimers (excluding dimers).   The ephrin-A multimer is an ephrin-A multimer obtained by multimerizing an ephrinA-Fc fusion protein in which an IgG (Fc) fragment is fused to the C-terminus of the ephrin-A extracellular domain. A therapeutic agent for central nervous system disorder for ventricular or striatum injection.   The ventricle or the ventricle according to claim 2, wherein the ephrin-A multimer is a multimer obtained by multimerizing an ephrin-A fusion protein in which the TSP5 coiled-coil domain is fused to the C-terminus of the ephrin-A extracellular domain. A therapeutic agent for central nervous system disorder for striatum injection.   The brain according to claim 4, wherein the ephrin-A multimer is a multimer obtained by multimerizing an ephrin-A fusion protein in which a plurality of TSP5 coiled-coil domains are fused to the C-terminus of the ephrin-A extracellular domain. A therapeutic agent for central nervous system disorder for room or striatum injection.   The ephrin-A is ephrin-A1, The therapeutic agent for central nervous system disorder for ventricular or striatum injection according to any one of claims 2 to 5.   The therapeutic agent for central nervous system disorder for ventricular or striatum injection according to any one of claims 1 to 6, wherein Parkinson's disease is to be treated.   A method for treating a central nervous disorder, which comprises injecting a ligand that binds to EphA and stimulates signal transduction from EphA into the ventricle or striatum to stimulate signal transduction from EphA.   A method for treating central nervous disorder, characterized by injecting ephrin-A multimers (excluding dimers) into the ventricle or striatum.   ephrin-A multimer in which ephrinA-Fc fusion protein with IgG (Fc) fragment fused to C-terminal of ephrin-A extracellular domain is injected into the ventricle or striatum How to treat neuropathy.   Injecting ephrin-A multimers into the cerebral ventricle or striatum, which is a multimerized ephrinA fusion protein in which the TSP5coiled-coil domain is fused to the C-terminus of the ephrin-A extracellular domain Method of treatment.   It is characterized by injecting into the ventricle or striatum an ephrin-A multimer that is a multimerized ephrin-A fusion protein in which multiple TSP5coiled-coil domains are fused to the C-terminal side of the ephrin-A extracellular domain. To treat CNS disorders.   The method for treating central nervous system disorder according to any one of claims 9 to 12, wherein the ephrin-A multimer is injected once, intermittently or continuously.   The method for treating central nervous system disorder according to any one of claims 8 to 13, wherein Parkinson's disease is a treatment target.   A nucleic acid having the base sequence represented by SEQ ID NO: 1.   A nucleic acid having the base sequence represented by SEQ ID NO: 2.   An ephrin-A multimer expressed from the nucleotide sequence represented by SEQ ID NO: 1.   An ephrin-A multimer expressed from the nucleotide sequence represented by SEQ ID NO: 2.