JPWO2005082135A1 - Axon regeneration promoter - Google Patents

Abstract

An axonal regeneration promoter containing a conventional protein kinase C inhibitor as an active ingredient is disclosed. The axonal regeneration-promoting agent of the present invention is effective for the regeneration of central nerve axons, and contributes to the treatment of patients who are damaged in the central nervous system such as the spinal cord due to traffic accidents or cerebrovascular disorders.

Description

  The present invention relates to an axonal regeneration promoting agent capable of promoting regeneration of nerve cells, particularly nerve cells of the central nervous system.

  If the central nervous system such as the spinal cord is damaged by a traffic accident or other injury or cerebrovascular disorder, the nerve function is lost and cannot be regenerated. In contrast to the regeneration of peripheral nerves. Because the central nervous system cannot be regenerated when damaged, damage to the central nervous system often results in partial or complete paralysis. Therefore, regenerating damaged central nerves is an important issue in the medical field.

  However, since the axons of the adult central nervous system can be regenerated through a peripheral nerve graft (Non-patent Document 1), the main cause of the adult central nervous system not regenerating is the local environment surrounding nerve cells. It is suggested that So far, Nogo, myelin-binding glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp) have been identified as three main inhibitors that inhibit central nerve regeneration. Nogo was identified as the corresponding antigen of monoclonal antibody IN-1 (Non-patent Documents 2 to 4). MAG, which is known to play an important role in the formation and maintenance of the myelin sheath (Non-Patent Documents 5 to 8), was found to inhibit axonal growth from certain neurons (non- Patent Documents 9 and 10). OMgp (Non-Patent Document 11), the main peanut agglutinin-binding polypeptide in the white matter of the adult central nervous system, has been identified as a third inhibitor of axonal growth (Non-Patent Documents 12 and 13). Nogo, MAG and OMgp are known to bind to NgR with p75 as a co-receptor, suggesting that they share a common signal transduction pathway (Non-patent Document 14). To 17).

  It has been studied to regenerate axons by eliminating or inhibiting these inhibitors. However, studies using mice that knocked out each of these inhibitors revealed that central axons cannot be regenerated just by eliminating these inhibitors (Non-Patent Documents 18 to 22). Furthermore, it has been reported that even when functional p75NTR is depleted or soluble p75N-Fc is administered, regeneration of the damaged spinal cord is not promoted (Non-patent Document 23).

[Non-Patent Document 1] S. David, AJ Aguayo, Science 214, 931-3 (Nov 20, 1981).
[Non-Patent Document 2] MS Chen et al., Nature 403, 434-9 (Jan 27, 2000).
[Non-Patent Document 3] T. GrandPre, F. Nakamura, T. Vartanian, SM Strittmatter, Nature 403, 439-44 (Jan 27, 2000).
[Non-Patent Document 4] P. Caroni, ME Schwab, Neuron 1, 85-96 (Mar, 1988).
[Non-Patent Document 5] S. Carenini, D. Montag, H. Cremer, M. Schachner, R. Martini, Cell Tissue Res 287, 3-9 (Jan, 1997).
[Non-Patent Document 6] M. Fruttiger, D. Montag, M. Schachner, R. Martini, Eur J Neurosci 7, 511-5 (Mar 1, 1995).
[Non-Patent Document 7] N. Fujita et al., J Neurosci 18, 1970-8 (Mar 15, 1998).
[Non-Patent Document 8] J. Marcus, JL Dupree, B. Popko, J Cell Biol 156, 567-77
(Feb 4, 2002).
[Non-patent document 9] G. Mukhopadhyay, P. Doherty, FS Walsh, PR Crocker, MT Filbin, Neuron 13, 757-67 (Sep, 1994).
[Non-Patent Document 10] L. McKerracher et al., Neuron 13, 805-11 (Oct, 1994).
[Non-Patent Document 11] DD Mikol, K. Stefansson, J Cell Biol 106, 1273-9 (Apr, 1988).
[Non-Patent Document 12] V. Kottis et al., J Neurochem 82, 1566-9 (Sep, 2002).
[Non-patent document 13] KC Wang et al., Nature 417, 941-4 (Jun 27, 2002).
[Non-Patent Document 14] KC Wang, JA Kim, R. Sivasankaran, R. Segal, Z. He,
Nature 420, 74-8 (Nov 7, 2002).
[Non-patent Document 15] M. Domeniconi et al., Neuron 35, 283-90 (Jul 18, 2002).
[Non-Patent Document 16] AE Fournier, T. GrandPre, SM Strittmatter, Nature 409, 341-6 (Jan 18, 2001).
[Non-patent document 17] T. Yamashita, H. Higuchi, M. Tohyama, J Cell Biol 157, 565-70 (May 13, 2002).
[Non-patent document 18] U. Bartsch et al., Neuron 15, 1375-81 (Dec, 1995).
[Non-patent document 19] CJ Woolf, Neuron 38, 153-6 (Apr 24, 2003).
[Non-patent Document 20] JE Kim, S. Li, T. GrandPre, D. Qiu, SM Strittmatter, Neuron 38, 187-99 (Apr 24, 2003).
[Non-patent document 21] B. Zheng et al., Neuron 38, 213-24 (Apr 24, 2003).
[Non-Patent Document 22] M. Simonen et al., Neuron 38, 201-11 (Apr 24, 2003).
[Non-Patent Document 23] X. Song et al., J Neurosci 24, 542-6 (Jan 14, 2004).
[Non-patent Document 24] Lin, WW, Wang, CW & Chuang, DMJ Neurochem. 68, 2577-2586 (1997).
[Non-patent Document 25] Eichholtz, T., et al. 1993. J. Biol. Chem. 28, 1982; Ward, NE, and OAfBrian, CA 1993. Biochemistry 32, 11903
[Non-patent Document 26] Martiny-Baron, G., et al. (1993) J. Biol. Chem. 268: 9194-9197
[Non-patent Document 27] Seewald, S. et al. (1999) Am. J. Hypertens. 12: 532-537
[Non-patent Document 28] Sveltov, S., and Nigami, S. 1993. Biochim. Biophys. Acta
1177, 75
[Non-patent document 29] Ku, W.-C., et al. 1997. Biochem. Biophys. Res. Commun. 241, 730
[Non-patent Document 30] Doherty, P. et al., 1990. Nature 343: 464-466

  An object of the present invention is to provide a novel axonal regeneration promoting agent capable of promoting regeneration of central nerve axons.

  As a result of intensive studies, the inventors of the present application have found that MAG and Nogo, which have been considered to be substances that inhibit axonal regeneration, are axon reversal when the function of conventional protein kinase C is inhibited. The present inventors have found that it has a function of promoting regeneration, found that a conventional protein kinase C inhibitor is effective in promoting regeneration of axons, and completed the present invention.

  That is, the present invention provides an axonal regeneration promoter containing a conventional protein kinase C inhibitor as an active ingredient. Preferably, the axon is a central nervous system axon. Preferably, the conventional protein kinase C is protein kinase C-α, β1, β2, γ or υ. Particularly preferably, the conventional protein kinase C inhibitor is a cell-permeable PKC inhibitor 20-28 or Go6976.

  Also preferably, the axonal regeneration promoter of the present invention is a myelin-binding glycoprotein, Nogo peptide or myelin, or a variant thereof, and in a state where conventional protein kinase C is inhibited by the inhibitor, It further includes a protein that exhibits a regeneration promoting effect, or a fusion protein that includes these or the above-described mutants and that exhibits axon regeneration promoting effect. More preferably, the axonal regeneration promoter of the present invention includes myelin-binding glycoprotein, Nogo peptide or myelin, or a fusion protein containing these.

  In another aspect, the present invention relates to a method for identifying a candidate substance for an axon regeneration promoter, comprising contacting a test substance with conventional PKC and determining whether the test substance inhibits the function of conventional PKC. A method comprising determining is provided.

  ADVANTAGE OF THE INVENTION According to this invention, the novel axon regeneration promoter which can accelerate | stimulate the reproduction | regeneration of the axon of a central nerve was provided. The axonal regeneration promoting agent of the present invention is effective for the regeneration of central nerve axons, and is expected to greatly contribute to the treatment of patients with damage to the central nervous system such as spinal cord.

FIG. 1 shows the relative activity of PKC when cultured cerebellar granule neurons are treated with MAG for 10 minutes, 30 minutes or 1 hour. PKC activity is expressed as the amount of phosphorylated PKC-α relative to the amount of PKC-α. Relative values are given as a ratio to PKC activity at time 0. The results show the mean ± standard deviation in 3 experiments. The asterisk indicates that it is statistically significant (p <0.01 in Student's t test). FIG. 2 shows the axon length of cerebellar granule neurons in the presence of the components shown. In the presence of PKCI, MAG-Fc and Nogo peptides stimulated axon growth, respectively. Data show mean ± standard deviation. The asterisk indicates that it is statistically significant (p <0.01 in Student's t test). FIG. 3 shows the results of the growth cone collapse assay. Data show mean ± standard deviation. The asterisk indicates that it is statistically significant (p <0.01 in Student's t test). FIG. 4 shows the results of affinity precipitation of RhoA in cerebellar granule neurons. MAG-Fc and Nogo peptide activated RhoA in the presence or absence of PKCI.

  The axonal regeneration promoter of the present invention contains a conventional protein kinase C inhibitor as an active ingredient (hereinafter, “protein kinase C” may be abbreviated as “PKC”). As conventional PKCs, PKC-α, PKC-β1, PKC-β2, and PKC-γ (all of which are non-patent documents 24) are known. Inhibitors of PKC-ζ and PKC-λ, which are atypical PKCs other than conventional PKC, do not have an axonal regeneration promoting effect.

  Various inhibitors of conventional PKC (hereinafter sometimes abbreviated as “PKCI”) are known, and any of them can be used, but those having high cell membrane permeability are preferred. Known PKCIs that can be used in the present invention include cell-permeable PKC inhibitor 20-28 (the cell permeable PKC inhibitor 20-28, non-patent document 25), Go 6976 (non-patent document 26), cell permeability. Examples include PKC inhibitor 19-27 (the cell permeable PKC inhibitor 19-27, Non-Patent Document 27), calphostin C (Calphostin C, Non-Patent Document 28), and GF 109203X (Non-Patent Document 29). Of these, the cell permeable PKC inhibitor 20-28, Go 6976 and the cell permeable PKC inhibitor 19-27 are preferred, and the cell permeable PKC inhibitor 20-28 and Go 6976 are particularly preferred. These PKCIs can be used alone or in combination of two or more.

The structures of the cell permeable PKC inhibitor 20-28, Go 6976 and the cell permeable PKC inhibitor 19-27 are as follows.
the cell permeable PKC inhibitor 20-28
N-Myr-Phe-Ala-Arg-Arg-Lys-Gly-Ala-Leu-Arg-Gln-NH ₂ (SEQ ID NO: 5)
(However, Myr is amino-terminal myristoylation)
Go 6976 C24H18N4O
12- (2-Cyanoethyl) -6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole
the cell permeable PKC inhibitor 19-27
Myr-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-OH (SEQ ID NO: 6)
(However, Myr is amino-terminal myristoylation)

  Moreover, the axonal regeneration promoter of the present invention may contain a monoclonal antibody that can bind to conventional PKC and inhibit its function as a conventional protein kinase C inhibitor. To obtain such monoclonal antibodies, according to methods well known in the art, an animal is immunized using conventional PKC as a sensitizing antigen, the resulting immune cells are removed and fused with myeloma cells, The hybridoma producing the antibody is cloned. The monoclonal antibody can be prepared from the supernatant of the culture solution obtained by culturing the hybridoma or from the ascites of the mouse after intraperitoneal administration of the hybridoma to the mouse.

  Furthermore, in addition to antibodies produced by hybridomas, recombinant antibodies, chimeric antibodies, CDR-grafted antibodies, fragments of these antibodies, and the like can also be used as conventional PKC inhibitors in the present invention.

  Recombinant antibodies are obtained by cloning cDNA encoding an antibody from a hybridoma that produces a monoclonal antibody that binds to a conventional PKC, inserting this into an expression vector, and transforming animal cells, plant cells, etc. It can be produced by culturing the body.

  A chimeric antibody is composed of a heavy chain variable region and a light chain variable region of an antibody derived from a certain animal (eg, mouse), and a heavy chain constant region and a light chain constant region of an antibody derived from another animal (eg, human). Antibody. A chimeric antibody is one in which a cDNA encoding the heavy chain variable region and light chain variable region of an antibody is cloned from a hybridoma producing a monoclonal antibody that binds to a conventional PKC, while the heavy chain constant region of an antibody derived from another animal. And a cDNA encoding the light chain constant region can be cloned, inserted into a suitable expression vector, and expressed in a host cell for recombinant production.

  A CDR-grafted antibody is an antibody in which the complementarity determining region (CDR) of an antibody from one animal (eg, mouse) is transplanted to the complementarity determining region of an antibody from another animal (eg, human). The gene encoding the CDR-grafted antibody is designed based on the gene sequence of the heavy chain variable region and the light chain variable region of the antibody cloned from the hybridoma producing the monoclonal antibody that binds to the conventional PKC. It can be obtained by substituting the corresponding CDR sequences in vectors containing genes encoding heavy and light chain variable regions of antibodies derived from other animals.

  Examples of antibody fragments that can bind to conventional PKC include Fab, F (ab ′) 2, Fab ′, scFv, and diabody. These antibody fragments are obtained by treating the above-described anti-conventional PKC monoclonal antibody of the present invention with an enzyme such as papain or trypsin, or introducing an expression vector incorporating a gene encoding them into a host cell. It can be manufactured by obtaining.

  Furthermore, the axonal regeneration promoter of the present invention includes, as a conventional protein kinase C inhibitor, an antisense oligonucleotide, a ribozyme, a molecule that causes RNA interference (RNAi) (for example, dsRNA, siRNA, shRNA, miRNA) and the like. It may be. These nucleic acids can bind to and inhibit the expression of a conventional PKC gene or mRNA encoding a conventional PKC. General methods for controlling gene expression using antisense, ribozyme technology and RNAi technology, or gene therapy methods for expressing exogenous genes in this manner are well known in the art.

  An antisense oligonucleotide represents a nucleic acid molecule having a sequence complementary to mRNA encoding conventional PKC or a derivative thereof. Antisense oligonucleotides specifically hybridize with mRNA and inhibit protein expression by inhibiting transcription and / or translation. Binding may be by Watson-Crick or Hoogsteen-type base pair complementarity, or by triplex formation.

  A ribozyme refers to the RNA structure of one or more RNAs that have catalytic properties. Ribozymes generally exhibit endonuclease, ligase or polymerase activity. Various ribozymes of secondary structure, such as hammerhead type and hairpin type ribozymes are known. RNA interference (RNAi) refers to a technique of silencing a target gene using a double-stranded RNA molecule.

  The route of administration of the axonal regeneration-promoting agent of the present invention is preferably parenteral, and in particular, it is preferably injected directly into the nerve damage site.

  The dose is appropriately selected according to the type of PKCI, the route of administration, the degree of nerve damage, etc., but when administered directly to the injured site, the dose of PKCI per injured site is 1 day for adults. In general, it is 0.01 mg to 1 mg, preferably 0.05 mg to 0.5 mg. For parenteral administration other than direct administration, such as intravenous injection, it is usually about 10 times the above dose.

  PKCI can be administered as is, but is usually formulated using a carrier used in medicine. Any carrier commonly used in the pharmaceutical field can be used as the carrier for the preparation. For example, physiological saline or phosphate buffered saline is preferably used for preparing an injection. Further, commonly used additives such as emulsifiers and osmotic pressure regulators may be included.

  The axonal regeneration-promoting agent of the present invention is effective for promoting regeneration of nerve cells, particularly nerve cells of the central nervous system such as spinal cord. Therefore, it can be used for treatment of central nerve damage caused by accidents. In the following examples, mouse neurons were taken out and tested, so experiments were performed with MAG, Nogo, or myelin added. In vivo nerve fibers, these are contained in the nerve sheath. Therefore, it is not necessary to add MAG, Nogo or myelin separately to the axonal regeneration promoter.

  However, if desired, MAG, Nogo or myelin may be added separately to the axonal regeneration promoter. MAG, Nogo, and myelin are well known and readily available. The nucleotide sequence of the human MAG gene and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 1 and 2 (GenBank Accession No. AC002132). In addition, the nucleotide sequence of human Nogo gene and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 3 and 4, respectively (GenBank Accession No. AY102279). The gene sequences and amino acid sequences of MAG and Nogo of mammals other than humans are also known. For example, those for rats are described in GenBank Accession No. NM_017190, and those for mice are described in GenBank Accession No. AY102284. Since myelin is a structure containing MAG, Nogo and other proteins, there is no corresponding myelin amino acid sequence. In addition, a plurality of natural mutants of human MAG are known (GenBank Accession No. BC053347), and such natural mutants may be used. MAG, Nogo and myelin can be used alone or in combination of two or more.

  Furthermore, in general, a polypeptide having physiological activity has a physiological activity even if it is a mutant in which a small number of amino acids constituting the amino acid sequence are substituted or deleted, or a small number of amino acids are inserted. It is widely known that activity may be maintained. When MAG, Nogo, or myelin is added to the axon regeneration promoter of the present invention, these mutants exhibit an effect of promoting axon regeneration in a state where PKC is inhibited by the inhibitor. It may be added. Such a mutant preferably has an amino acid sequence having an identity of 80% or more, preferably 90% or more, more preferably 95% or more with the amino acid sequence of a natural polypeptide. The number of amino acids lost or inserted is preferably 1 to several. In addition, amino acid sequence identity can be easily performed using BLAST, which is a software widely used in this field. BLAST is available to anyone on the NCBI (National Center for Biotechnology Information) homepage and can be easily checked for identity using default parameters. Twenty kinds of amino acids constituting natural proteins are neutral amino acids having low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids having hydrophilic side chains (Asn , Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp) Can be grouped into When obtaining a mutant in which a small number of amino acids are substituted, the physiological activity of the polypeptide is often not changed by substitution within each of these groups. In addition, since fusion proteins containing MAG, Nogo, myelin, or the above-mentioned mutants usually maintain the physiological activity of these polypeptides, such fusion proteins can also be used. For example, in the following examples, a fusion protein of MAG and the Fc region of immunoglobulin is used, but such a fusion protein may be added to the axonal regeneration promoter of the present invention.

  When MAG, Nogo or myelin is added, a natural protein derived from the animal species to be administered (meaning that it does not contain mutation (but may be produced by genetic engineering techniques)) or a fusion protein containing it Particularly preferred are natural ones.

  The concentration of MAG of the axonal regeneration promoter is usually 0 to 100 μM, preferably 0 to 50 μM, the concentration of Nogo is usually 0 to 10 μM, preferably 0 to 0.5 μM, and the concentration of myelin is Usually, it is 0-100 ng / μl, preferably 0-10 ng / μl.

  In another aspect, the present invention provides a method for identifying a candidate substance for an axonal regeneration promoter. The method includes contacting a test substance with a conventional PKC and determining whether the test substance inhibits the function of the conventional PKC. Conventional PKC functions include both the enzymatic activity of conventional PKC to phosphorylate its substrate protein and the ability of conventional PKC to bind ligands.

  The enzymatic activity of conventional PKC can be readily measured using any of a variety of kinase activity assays known in the art. By comparing the kinase activity in the presence and absence of the test substance, it can be determined whether the test substance inhibits the enzymatic activity of conventional PKC. Also, the ability of conventional PKC to bind to its corresponding ligand can be determined by binding assays well known to those skilled in the art, such as, but not limited to, gel shift assays, radiolabel competition assays, chromatographic fractionation, etc. .

  Substances identified by these assays as inhibiting the function of conventional PKC are considered candidates for axon regeneration promoters. The candidate substance is then promoted to axon regeneration by measuring and comparing the growth of neuronal axons in the presence and absence of the candidate substance using assay methods known in the art. It can be determined whether or not there is an effect.

  The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, the contents described in the specification and drawings of Japanese Patent Application No. 2004-51157, which is the application on which the priority of the present application is based, are cited herein as a part of this specification.

  The present invention will be described in more detail with reference to the following examples, but these examples do not limit the scope of the present invention.

Materials and Methods Measurement of PKC activity PKC assay was performed using a PepTag assay kit (trade name, Promega, Madison, Wis., USA), which is a PKC-based detection kit that does not use radioactivity. Serum-starved cultured cerebellar granule cells were obtained from MAG-Fc (a fusion protein of MAG and immunoglobulin Fc region, available from Sigma, St. Louis, MO) (25 μg / ml) and Nogo peptide (Texas, USA). San Antonio's Alpha Diagnostic (4 μM) was applied. Each sample was incubated with PepTag C1 peptide (2 μg), which is a substrate for PKC, at 30 ° C. for 30 minutes. Samples were separated on a 0.8% agarose gel at 100 V for 15 minutes. The phosphorylated peptide substrate moved to the anode (+), and the non-phosphorylated peptide substrate moved to the cathode (-). The gel was photographed on a transilluminator.

  Phosphate-specific antibodies were used for the measurement of phosphorylated PKC. 10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10% glycerol, 100 μM sodium orthovanadate, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM EGTA and protease inhibitor mixture (Roche Biochemicals) Cells were lysed with 0.1% NP-40 (trade name, nonionic surfactant) containing The cell lysate was subjected to SDS-PAGE and Western blot analysis was performed by a conventional method. Polyclonal anti-phospho PKC-α (Ser657 phosphorylated, Upstate Biotech, Charlottesville, VA), polyclonal anti-phospho PKC-ζ / λ (Cell Signaling Tech) and polyclonal anti-PKC-α antibody (Santa Cruz Biotech) Made).

2. Axon Growth Assay To prepare cerebellar neurons, cerebellums from two 7-day-old rats were made one in 5 ml 0.025% trypsin solution, crushed and incubated at 37 ° C. for 10 minutes. Cells were collected by adding DMEM medium containing 10% FCS and centrifuging at 800 rpm. Neurons were plated in Sato medium (30) on chamber slides coated with poly-L-lysine. For growth assays, plated cells were incubated for 24 hours, fixed with 4% (w / v) paraformaldehyde, and monoclonal antibodies (TuJ1, Covance Research for the purpose of recognizing neuron-specific β-tubulin III protein. Products, Inc., Denver, USA). The length of the longest process (axon) or the total neurite length (process outgrowth) of β-tubulin III positive neurons was measured. In each experiment, at least 100 axon lengths were measured and the same experiment was repeated three times. As described in the result column, MAG-Fc (25 μg / ml), Nogo peptide (4 μM), the cell permeable PKC inhibitor 20-28 (2 μM, Calbiochem) or Go6976 (0.1 μM, Biosource) , Added to the medium after the plate.

3. Growth cone collapse assay
Incubate explants of embryonic day 12 chicken dorsal root ganglia on plastic slides pre-coated with 100 μg / ml poly-L-lysine and extract soluble central nervous system myelin at the concentrations indicated in the results column (Sigma), MAG-Fc (25 μg / ml) or Nogo peptide (4 μM) for 30 minutes. Explants were fixed with 4% (w / v) paraformaldehyde and stained with fluorescently labeled phalloidin (phalloidin (Sigma)). In each experiment, at least 100 growth cones were examined and the same experiment was repeated three times.

4). Affinity precipitation of GTP-RhoA
50 mM Tris (pH 7.5) containing 1% Triton X-100 (trade name), 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl and 10 mM MgCl ₂ , 10 μg / ml leupeptin and 10 μg / ml aprotinin Cells were lysed with. The cell lysate was clarified by centrifugation at 13000 g, 4 ° C. for 10 minutes, and the supernatant was incubated for 45 minutes at 4 ° C. with 20 μg GST-Rho binding region of Rhotekin beads (trade name, manufactured by Upstate Biotech). The beads were washed four times with a washing buffer (1% Triton X-100 (trade name), 150 mM NaCl, 10 mM MgCl ₂ , 50 mM Tris (pH 7.5) each containing 10 μg / ml leupeptin and aprotinin). Bound Rho was detected by Western blot using a monoclonal antibody against RhoA (Santa Cruz Biotech, Santa Cruz, California, USA).

Result 1. Measurement of PKC activity When cultured granule cells were treated with 25 μg / ml MAG-Fc or 4 μM Nogo peptide for 5 minutes, PKC activity was clearly increased (a thick band of phosphorylated PKC was observed in the agarose electrophoresis). ). Furthermore, PKC activity increased significantly when treated with MAG-Fc for 10 minutes, 30 minutes, or 1 hour (FIG. 1). PKC-α, β1, β2, and γ are expressed as conventional PKC in postnatal cerebellar granule neurons. Western blot analysis showed that PKC-α Ser657 phosphorylation by MAG-Fc was significantly increased, but PKC-α protein concentration did not change significantly. However, PKC-ζ / λ, an atypical PKC, was not phosphorylated by MAG in experiments using phosphate-specific antibodies (data not shown).

2. Axon Growth Assay Next, we examined whether PKC or MAG was related to the effects of MAG or Nogo on axon growth. MAG-Fc (25 μg / ml) or Nogo peptide (4 μM) inhibited axon growth of cerebellar granule neurons from 7-day-old rats (FIG. 2). Surprisingly, however, MAG-Fc and Nogo peptides are PKC-α and -β inhibitors, the cell permeable PKC inhibitor 20-28 (in FIG. 2, “PKCI In the presence of “)”, it dramatically stimulated axon growth (Fig. 2), but PKCI itself had no axon growth effect (Fig. 2) .MAG-Fc or Nogo The degree of axonal growth induced by A was about twice that of controls, and similar results were obtained with Go6976, an inhibitor of PKC-α, β and υ (Figure 2). However, this effect was not obtained with HA-1077 (10 mM), a Rho kinase inhibitor (Figure 2), and PKC activity was measured by the cell permeable PKC inhibitor 20-28 (the cell permeable PKC inhibitor). 20-28) or inhibited by Go6976 (data not shown) These results depended on the activity of conventional PKC by MAG and Nogo It shows the bi-directional control of axon growth.

3. Growth cone collapse assay To examine the effects of MAG and Nogo on the nerve growth cone, embryonic day 12 chicken DRG (dorsal root ganglion) explants were used. When MAG-Fc (25 μg / ml) or Nogo peptide (4 μM) was administered in a bath (bath application), it showed significant growth cone collapse activity (FIG. 3). Similar to the results of the axonal growth assay, MAG-Fc and Nogo peptides promoted growth cone extension in the presence of PKCI compared to controls. Myelin purified from bovine white matter caused growth cone collapse at concentrations of 0.1-10 ng / μl, whereas PKCI completely reversed the effect mediated by myelin (FIG. 3). These findings indicate that MAG, Nogo, and myelin inhibit axon growth by activating PKC and cause growth cone collapse, but PKCI-induced axon growth promotion and growth cone extension were independent of PKC Suggests that it is mediated by a mechanism.

4). Affinity precipitation of GTP-RhoA
A possible mechanism by which MAG or Nogo-induced axonal regeneration is converted from inhibition to promotion is that PKC modulates Rho activity. This is because Rho has been shown to be a key signal molecule in axonal elongation inhibition (Non-patent Documents 17 and 14). Therefore, we measured RhoA activity in neurons. When MAG-Fc or Nogo was added to cerebellar neurons of 7-day-old rats for 30 minutes, RhoA was activated (FIG. 4). PKCI had no effect on Rho activity induced by MAG-Fc or Nogo peptide. Thus, the promotion of cerebellar neuron axon growth by the effect of inhibiting PKC is not mediated by blocking RhoA activity, indicating that conventional PKC is not upstream of RhoA. .

  The axonal regeneration promoting agent of the present invention is effective for regeneration of an injured central nerve, and is useful as a therapeutic agent for a patient who has injured the central nervous system.

Claims (7)

  An axonal regeneration promoter containing a conventional protein kinase C inhibitor as an active ingredient.   The axonal regeneration promoter according to claim 1, wherein the conventional protein kinase C is protein kinase C-α, β1, β2, γ or υ.   The axon regeneration promoter according to claim 1 or 2, wherein the axon is a central axon.   The axonal regeneration promoter according to any one of claims 1 to 3, wherein the conventional protein kinase C inhibitor is a cell-permeable PKC inhibitor 20-28 or Go6976.   Myelin-binding glycoprotein, Nogo peptide or myelin, or a variant thereof that exhibits an axon regeneration promoting effect in a state where conventional protein kinase C is inhibited by the inhibitor, or these or the above The axon regeneration promoting agent according to any one of claims 1 to 4, further comprising a fusion protein containing a mutant that exhibits an axon regeneration promoting effect.   The axon regeneration promoter according to claim 5, comprising a myelin-binding glycoprotein, a Nogo peptide, myelin, or a fusion protein containing these. A method for identifying a candidate substance for an axonal regeneration promoter, comprising contacting a test substance with a conventional PKC and determining whether the test substance inhibits the function of the conventional PKC.