WO2006016429A1

WO2006016429A1 - Axon formation and elongation employing nerve growth cone localized molecule shootin1 or its splicing variants, and applications for nerve regeneration

Info

Publication number: WO2006016429A1
Application number: PCT/JP2005/002534
Authority: WO
Inventors: Naoyuki Inagaki; Tadayuki Shimada; Michinori Toriyama; Osamu Ohara; Takahiro Nagase
Original assignee: National University Corporation NARA Institute of Science and Technology; Kazusa Dna Research Institute
Priority date: 2004-08-13
Filing date: 2005-02-10
Publication date: 2006-02-16
Also published as: JP4592352B2; JP2006050980A

Abstract

A novel molecule Shootin1 was identified, whose expression changes before and after formation of polarity in nerve cells. Shootin1 is localized in the growth cone of the axon tip, which is crucial for axon formation and elongation. Shootin1 is expressed specifically in the brain and its expression significantly increases during periods when axon formation is active in individuals. Shootin1 was found to be present at high concentrations in the growth cone of axon tips, and when expressed exogenously in cultured nerve cells, Shootin1 induced formation of a plurality of axons. Because Shootin1 has such axon formation inducing function, it is possible to induce and accelerate axon formation and elongation in nerve cells, by positively controlling the expression or activity of Shootin1 or its splicing variants.

Description

DESCRIPTION

AXON FORMATION AND ELONGATION EMPLOYING NERVE GROWTH CONE LOCALIZED MOLECULE SHOOTINl OR ITS SPLICING VARIANTS, AND APPLICATIONS FOR NERVE REGENERATION

Technical Field

The present invention relates to methods for inducing and promoting the formation or elongation of nerve cell axons using nerve growth cone localized molecule Shootinl or splicing variants thereof. The present invention in particular contributes to the development of novel nerve regeneration technology, and can be employed in research and establishment of axon regeneration medical technologies, such as an effective remedy for damages in central and peripheral nerves caused by stroke or spinal cord injury, for example.

Background Art

Nerve cells (= Neurons) have polarity, in other words, directionality. More concretely, they have a plurality of dendrites and a single axon and receive a variety of information from other nerve cells at their dendrites and integrate their inputs within their cell body to convert them into an action potential that transmits over their axon from the cell body to the synaptic terminus. Neurotransmitters are then released from the synaptic terminus to transmit the information to a target cell. This phenomenon is the basis for high-order life activities such as memory, learning, and exercise in higher organisms. Much of the molecular mechanisms of the polarity formation and maintenance in nerve cells, however, remains unknown (for research regarding polarity formation, see for example Dotti, C. G., Sullivan, C. A., Banker, G. A. (1988) "The establishment of polarity by hippocampal neurons in culture." J. Neurosci. 8, 1454-1468). Deciphering the molecular mechanisms of the polarity formation in nerve cells would make clear the molecular mechanisms of axon formation and elongation during developmental stages, and also formation of neural networks. The axon formation is one aspect of the polarity formation in nerve cells, and thus identification of the molecules involved in the polarity formation provides molecular tools which could be employed to form or elongate axons. There is also a possibility that those same molecules could be utilized in the development of a novel nerve regeneration technology, such as use in the development of medical treatment for the regeneration of severed or degenerated axons. Developing such medical treatment is of great importance. For example, there are no effective drug therapies for axon regeneration in injuries in central and peripheral nerves caused by stroke or trauma, and currently the primary medical approach for such injuries is rehabilitation for the purpose of recovery of nerve function. In particular, the axons of central nerves cannot be regenerated once they have been damaged, and thus many patients are forced to live on a confined wheelchair through life, and such injuries place a very heavy burden on the patient, his family, and society.

DDBJ/EMBL/GenBank databases: accession number AK082304 discloses the cDNA sequence, and predicted amino acid sequence, of a gene cloned from the cerebellum library of a newborn mouse (PO). That data, however, do not provide any disclosure or indication of the function of the gene and its protein.

The first object of the present invention is to develop and provide a novel method for inducing and promoting axon formation and elongation in nerve cells, and for this object, to identify the molecule using proteomics analysis, such as two-dimensional electrophoresis and mass spectrometry, whose expression changes before and after the polarity formation in nerve cells and localized in the growth cone at the tip of the axon that is important for axon formation and elongation, and then analyze the function of such molecule. The second object of the invention is to provide a novel gene and a novel protein that can be used in the above method and utilized in nerve regeneration therapy for injuries in central nerves or peripheral nerves. The third object of the invention is to provide a method for screening therapeutic agents for nerve regeneration, and reagents for research, using the above molecule as a target or a probe. We used proteomics based on the high-sensitivity two-dimensional electrophoresis method that we developed (Inagaki N. and Katsuta K, Curr. Proteomics 1, 35-39, 2004) to screen the proteins in rat cultured hippocampal neurons, wholly analyzing the proteins whose expression increases in accordance with axon formation. The result was the identification of a novel protein (Shootinl) having a molecular weight of 52.6 kD. This molecule was analyzed further, leading to the findings that (1) it is expressed specifically in the brain and the amount of Shootinl expression exhibits a significant rise four days after birth, during which axons are formed, (2) Shootinl is highly concentrated in the growth cone at the axon tip, (3) exogenous expression of Shootinl in cultured nerve cells (that is, expression of the gene introduced from outside, not expression of the endogenous gene) induces the formation of a plurality of axons, (4) Shootinl is localized in the growth cone during axon formation and elongation, and exhibits dynamic movement within the growth cone, and (5) there are two splicing variants in addition to Shootinl, leading us to the present invention.

Disclosure of the Invention

The present invention includes the following industrially and medically useful inventions A) to O).

A) A method for inducing formation or elongation of axons in nerve cells by positively controlling (or regulating) an expression or activity of nerve growth cone localized molecule Shootinl or a splicing variant thereof. Here, the word "nerve cells" means "neurons."

Examples of methods for "positively controlling (or regulating) an expression or activity" of Shootinl, include (1) a method of introducing the Shootinl (or its splicing variant) gene into nerve cells to exogenously express Shootinl (or its splicing variant) and thereby increase expression within those cells, (2) a method of increasing expression of endogenous Shootinl (or its splicing variant), and (3) a method of increasing the activity of Shootinl (or its splicing variant), and combinations of these methods also are possible. Here "splicing variant" means a protein translated from mRNA formed by an alternative splicing. Examples of splicing variants of Shootinl include Shootin2 (p52b) and Shootin3 (p52c) discussed below, which have been determined by genome analysis.

B) The nerve axon formation/elongation inducing method set forth in A) above, characterized in that the expression of Shootinl or its splicing variant within nerve cells is positively controlled (or regulated) by introducing a gene for Shootinl or its splicing variant into those cells.

The gene can be introduced into nerve cells according to a normal method such as transfection of a recombinant expression vector into those cells. C) A nerve axon formation/elongation inducing agent, including a recombinant expression vector constructed such that a gene for nerve growth cone localized molecule Shootinl or a splicing variant thereof is expressed in nerve cells.

For the "recombinant expression vector", it is possible to use a viral vector, plasmid, phage, or cosmid, but there is no limitation to these.

Because there are a variety of promoters that function within a host cell, a promoter appropriate for the target cell can be selected and arranged at upstream of the Shootinl (or its splicing variant) gene.

D) The nerve axon formation/elongation inducing agent set forth in C) in which the recombinant expression vector is constructed so as to express the gene for human-, rat-, or mouse-derived Shootinl or a splicing variant thereof.

The cDNA sequences and amino acid sequences of human-, rat-, and mouse-derived Shootinl are shown in SEQ ID NOs. 1 to 3, 4 to 6, and 7 to 9, respectively, of the Sequence Listing, and the recombinant expression vector can be prepared based on these sequences.

E) A gene therapeutic agent for nerve regeneration, including a recombinant expression vector constructed such that a gene for nerve growth cone localized molecule Shootinl or a splicing variant thereof is expressed in nerve cells. This gene therapeutic agent can be used for axon regeneration in severed or degenerated nerve cells or neural tissue. F) The gene therapeutic agent for nerve regeneration set forth in E) in which the recombinant expression vector is constructed so as to express a gene for human-, rat-, or mouse-derived Shootinl or a splicing variant thereof.

G) A human-derived Shootinl protein, indicated by (a) or (b) below: (a) a protein having the amino acid sequence of SEQ ID NO. 3;

(b) a protein having an amino acid sequence obtained by the deletion, replacement, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO. 3, and having the activity of inducing axon formation or elongation in nerve cells. "Shootinl protein" in the present invention may have additive polypeptides. An example of such additive polypeptides includes a case of having an epitope such as His, Myc, or flag.

The "deletion, replacement, or addition of one or several amino acids" means the deletion, replacement, and/or addition of a number of amino acids that can be deleted, replaced, and/or added by a conventional method such as site-specific mutagenesis. That is, the protein of (b) is a mutant protein of the protein of (a), and here "mutant" is used primarily to mean mutation artificially introduced by a conventional method, but it also includes similar naturally-occurring mutant proteins isolated and purified. H) A rat-derived Shootinl protein of (a) or (b) below:

(a) a protein having the amino acid sequence of SEQ ID NO. 6;

(b) a protein having an amino acid sequence obtained by the deletion, replacement, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO. 6, and having the activity of inducing axon formation or elongation in nerve cells.

I) A Shootinl gene, encoding any one protein set forth in G) or H). Examples of the Shootinl gene include a gene having the sequence shown in SEQ ID NO. 1 or 4. "Gene" in the present invention is preferably cDNA, but it can also be RNA or genomic DNA. Furthermore, "gene" in the present invention may include a sequence other than coding region of the Shootinl protein, such as a sequence for untranslated region (UTR) or vector sequence (including expression vector sequence). J) A DNA of any one of (a) to (c) below:

(a) DNA having the sequence of SEQ ID NO. 1 or 4;

(b) DNA that has a sequence hybridized under stringent conditions to DNA having a sequence that is complementary to the sequence of SEQ ID NO.

1 or 4, and that encodes a protein having the activity of inducing axon formation or elongation in nerve cells;

(c) DNA that is obtained by screening an expression library using an anti-Shootinl antibody and that encodes a protein having the activity of inducing axon formation or elongation in nerve cells.

K) A method for screening therapeutic agents for nerve regeneration, including screening a compound that positively controls (or regulates) an expression or activity of nerve growth cone localized molecule Shootinl or a splicing variant thereof. Examples include (1) a method of screening compounds that bind

Shootinl (or its splicing variant), by binding assay such as affinity column, yeast-two-hybrid, and immunoprecipitation, and increase its activity, and (2) a method of screening compounds that when administered to nerve cells increase the expression of endogenous Shootinl (or its splicing variant). L) An antibody, which is a reagent for research relating to nerve axon formation or elongation or to nerve regeneration, and specifically detects Shootinl or a splicing variant thereof.

"Antibody" in the present invention is an antibody obtained by a known method, as a polyclonal antibody or monoclonal antibody, whose antigen is a Shootinl (or its splicing variant) protein or a peptide portion thereof.

M) The antibody of L), which is an anti-Shootinl antibody that detects human- or rat-derived Shootinl protein specifically.

N) The DNA shown in (c) among the DNAs of J), in which the DNA is obtained by screening an expression library using the antibody of L) or M). O) An RNAi reagent, which is a reagent for research relating to nerve axon formation or elongation or to nerve regeneration, and is introduced into cells in order to suppress specifically the expression of Shootinl or a splicing variant thereof.

The RNAi reagent can be siRNA (short interference RNA) or an RNAi expression vector. The siRNA and RNAi expression vectors can be designed using a known method based on the gene sequence of the suppression target, such as Shootinl. Further, the RNAi expression vector can be either (1) designed such that it expresses within a target cell as dsRNA that is single

RNA and has a hairpin structure of a suitable length, or (2) designed such that sense chain and antisense chain are each expressed within the target cell and become associated.

Shootinl, as described later, was found to have function that it induces formation of a plurality of axons when expressed exogenously in cultured nerve cells. Shootinl has the activity to induce axon formation or elongation, thus making possible application in the medical and pharmaceutical fields as a target molecule for axon regeneration treatment of central and peripheral nerves. Shootinl itself accelerates axon formation and thus by expressing Shootinl exogenously in the damaged nerve cells or neural tissue of a patient via a vector such as a virus, axon regeneration in those cells can be stimulated. Also, if such molecules that interact with Shootinl and activate endogenous Shootinl are found by the screening method of the present invention, then those molecules can become candidate molecules for axon regeneration therapy drugs. Furthermore, the human- and rat-derived novel Shootinl genes, Shootinl proteins, antibodies of these, and RNAi reagents can be used as reagents for research in nerve regeneration, for example. The splicing variants of Shootinl (Shootin2 and Shootin3, which are discussed later) are structurally similar to Shootinl as shown in FIG. 2 and because they have a common structure, they likely have a similar function and can be used similarly to Shootinl.

Brief Description of Drawings

FIG. 1 schematically shows the structure of Shootinl (p52a) made clear through analysis.

FIG. 2 compares the schematic structures of Shootinl (p52a) and its splicing variants Shootin2 (p52b) and Shootin3 (p52c). A-E in the figure correspond to the exons on the genome.

FIG. 3 shows the results of using anti-Shootinl antibody to examine the distribution of expression of Shootinl in various organs and to examine changes in the amount of Shootinl expression in the brain during developmental stages. FIG. 4 is a photograph showing the results of examining the distribution of Shootinl in cultured hippocampal neurons using anti-Shootinl antibody.

FIG. 5 is a photograph showing that overexpression of Shootinl leads to the formation of a plurality of axons in the cultured hippocampal neurons.

The two images are merged; one image of immunostaining using anti-myc antibody and another image of immunostaining using an axon marker antibody (anti-taul).

FIG. 6 is a photograph showing localization within cells when Shootinl is expressed exogenously in rat cultured hippocampal neurons, with the upper figure being a complete view of the nerve cells exogenously expressing Shootinl and the lower figure being a magnified view of the area around the axonal growth cone surrounded by a square.

Sequence Listing Free Text

SEQ ID NO. 1: cDNA sequence of human-derived Shootinl. SEQ ID NO. 2: cDNA sequence and amino acid sequence of human-derived

Shootinl.

SEQ ID NO. 3: Amino acid sequence of human-derived Shootinl.

SEQ ID NO. 4: cDNA sequence of rat-derived Shootinl.

SEQ ID NO. 5: cDNA sequence and amino acid sequence of rat-derived Shootinl.

SEQ ID NO. 6: Amino acid sequence of rat-derived Shootinl.

SEQ ID NO. 7: cDNA sequence of mouse-derived Shootinl.

SEQ ID NO. 8: cDNA sequence and amino acid sequence of mouse-derived

Shootinl. SEQ ID NO. 9: Amino acid sequence of mouse-derived Shootinl.

SEQ ID NO. 10: PCR forward primer sequence for cloning human cDNA. SEQ ID NO. 11: PCR reverse primer sequence for cloning human cDNA. SEQ ID NO. 12: PCR forward primer sequence for cloning rat cDNA. SEQ ID NO. 13: PCR reverse primer sequence for cloning rat cDNA.

Best Mode for Carrying Out the Invention

Specific embodiments of the present invention are described in detail below with reference to the drawings.

FIG. 1 shows the schematic structure of the Shootinl protein identified through analysis by the present inventors. The Shootinl protein has 456 amino acids in total and includes three Coiled-coil regions (C.Cl-3 in the drawing) and one Proline-rich-region. Firstly we called it ^Bp52a" because its molecular weight is 52.6 kD, but subsequent analysis revealed that the molecule moves like a shooting star at the tip of the nerve axon, and thus finally we named it "Shootinl." The Shootinl protein is not homologous with any known proteins, and from its genomic information we found that Shootinl homologs exist among rats, humans, Japanese macaque, mice, zebrafish, and blowfish.

SEQ ID NOs. 1 to 3 show the cDNA sequence and amino acid sequence of human-derived Shootinl determined by the present inventors. SEQ ID NOs. 4 to 6 show the cDNA sequence and amino acid sequence of rat-derived Shootinl determined by the present inventors. These genes and proteins are novel genes and proteins that have been cloned for the first time by the present inventors.

It should be noted that the gene of the present invention includes not only (1) the human-derived Shootinl gene having the base sequence shown in

SEQ ID NO. 1 and (2) the rat-derived Shootinl gene having the base sequence shown in SEQ ID NO. 4, but also (3) a gene that has a base sequence hybridized under stringent conditions to DNA having a base sequence that is complementary to the base sequence shown in SEQ ID NO. 1 or 4 and that encodes a protein that has the activity of promoting nerve axon formation (or elongation).

Hybridization under stringent conditions can be carried out using for example the method set forth in "Molecular Cloning: Cold Spring Harbor Laboratory Press, Current Protocols in Molecular Biology"; Wiley Interscience, and more specifically the following method is exemplified. DNA molecules from a cDNA library or the like are transferred to a membrane and hybridized with labeled probes within a hybridization buffer. The composition of the hybridization buffer is for example 0.1 wt% SDS, 5 wt% dextran sulfate, 1/20 content blocking reagent, and 2 to 7 x SSC. One example for the blocking reagent is to use a solution of 100 x Denhardt's solution, 2% (weight/volume) Bovine serum albumin, 2% (weight/volume) polyvinyl pyrrolidone prepared at five times concentration and then diluted to 1/20 concentration.

The hybridization temperature is in the range of 40 to 8O⁰C, more preferably 50 to 7O⁰C, and most preferably 55 to 65⁰C, and after incubation of between several hours and overnight, the membrane is washed with a washing buffer. The washing temperature is preferably room temperature or the temperature during hybridization. The washing buffer composition is preferably a 0.1 to 6 x SSC + 0.1 wt% SDS solution, and the membrane is washed several times with the washing buffer while altering the SSC concentration. The DNA hybridized with the probe is then detected using the label attached to the probe. SEQ ID NOs. 7 to 9 show the cDNA sequence and amino acid sequence of mouse-derived Shootinl. These sequences are disclosed in

DDBJ/EMBL/GenBank databases accession number AK082304, and are sequences that are already known. However, there has been no disclosure or suggestion regarding their function.

Analyzing the genome information revealed that in addition to Shootinl (p52a) there are two other splicing variants Shootin2 (p52b) and Shootin3 (p52c). FIG. 2 compares the overall structure of Shootinl (p52a in the drawing) with its splicing variants, Shootin2 (p52b in the drawing) and Shootin3 (p52c in the drawing). A to E correspond to the exons on the genome.

Mouse-derived Shootin2 (p52b) is constituted by 631 amino acids, and in common with Shootinl (p52a) in that it has regions corresponding to the exons A and B. On the other hand, rat-derived Shootin3 (p52c) is constituted by 599 amino acids, and in common with Shootinl (p52a) in that it has a region corresponding to the exon A. Each of these sequences is already known. The sequence of Shootin2, which is mouse-derived, is disclosed in accession number BC030338, and the sequence of Shootin3, which is rat-derived, is disclosed in accession number XM_214734. Shootin2 and Shootin3 exist in zebrafish, for example, in addition to mice and rats, and human homologs, too, likely exist. Shootin2 and Shootin3 are structurally similar to Shootinl, and thus conceivably have a similar function to Shootinl, that is, the activity of accelerating axon formation and elongation.

As regards Shootinl, cloned human Shootinl was expressed and based on this protein, anti-Shootinl antibody was created and analysis was performed, leading to the following findings (the experiment is later described in detail).

(1) Investigation of spatial and temporal expression of Shootinl in rat through Western blot analysis using the anti-Shootinl antibody showed that Shootinl was expressed specifically in the brain and its expression level was the strongest in the fourth day after birth (P4), when axon formation is active (FIG. 3).

(2) It was found that Shootinl was strongly condensed in the growth cone of elongating axon tips in cultured nerve cells prepared from hippocampus of rat embryo (E 18) (FIG. 4). (3) Exogenous expression of Shootinl in cultured hippocampal neurons induced the formation of a plurality of axons (FIG. 5). Shootinl showed localization toward the growth cone of the axon tip also when Shootinl was expressed exogenously in nerve cells (FIG. 6). (4) Shootinl was localized in the growth cone during formation and elongation of axons, and demonstrated dynamic movement in the growth cone.

These results show that Shootinl is localized in the growth cone of nerve axons and has an important role in their formation. In particular, the expression of Shootinl in nerve cells induces (promotes) axon formation, and thus by positively controlling (regulating) the expression or activity of Shootinl, it is possible to induce and accelerate axon formation or elongation in nerve cells.

One example is the method of introducing the Shootinl gene into nerve cells using a vector such as a viral vector to express Shootinl exogenously and induce and promote the formation or elongation of nerve axons. It is also possible to use this Shootinl gene vector in the regeneration of injured nerve cells as a gene therapy agent for nerve regeneration. Both in vivo and ex vivo introduction of the gene into the nerve cells are conceivable methods, and for efficient in vivo introduction of the gene into the nerve cells, it is possible to use a known gene carrier or drug delivery system.

Other examples of methods for positively controlling the expression or activity of Shootinl include the method of increasing the 'expression' of endogenous Shootinl and the method of increasing the 'activity' of endogenous

(or exogenous) Shootinl. These methods are preferably effected by administration of a drug, and thus a screening method for searching for compounds that increase Shootinl expression or activity is useful, and such a screening method also falls within the scope of the present invention.

Various types of conventional methods known for investigation of the expression level of genes and proteins and the change in protein activity, for example, can be adopted for the screening method of the present invention, and there are no particular limitations. It is also possible to use new screening methods that may be developed after the present invention. Both in vitro and in vivo screening methods are possible, as well as screening that is performed using a cell-free system. It is possible to use rat-, mouse-, and other animal derived Shootinl gene and protein in addition to those derived from humans. It is of course also possible to perform screening using information on three dimensional structure of the Shootinl protein.

Examples of the screening method include (1) screening methods for compounds that bind to Shootinl through a binding assay such as, affinity column, yeast-two-hybrid, and immunoprecipitation, and increase its activity, and (2) screening methods for compounds that when administered to nerve cells increase the expression of endogenous Shootinl.

As discussed above, it has been recognized that Shootinl is localized at a high concentration in nerve growth cones, and thus Shootinl molecules are likely bound to one another directly or indirectly via other molecules. If Shootinl does indeed have such a binding ability, then conceivably it can be activated and controlled depending on that binding. Shootinl is expressed at a high level in growth cones, which are exactly the sites where nerve cells elongate, and its expression promotes axon formation. Consequently, Shootinl is an important target molecule for medical regeneration of nerve axons. For the clinical use of Shootinl, it is preferable to make clear the intracellular molecular mechanism of the axon formation activity of Shootinl through further research. Anti-Shootinl antibodies that specifically detect the Shootinl protein and RNAi reagents that specifically suppress the Shootinl expression are useful as reagents for such research.

Anti-Shootinl antibody can be obtained as a polyclonal antibody through a known method by injecting the entire Shootinl protein or a peptide synthesized based on the epitope region thereof into a rabbit as an antigen. The epitope region can be deduced by predicting hydrophilic regions using the method of Kite & Doolittle, for example. Of course, it is also possible to create Shootinl monoclonal antibodies through a known method. Examples of known methods are those discussed in Antibodies: A laboratory manual (Cold Spring Harbor Laboratory, New York (1988)) by Harlow, et al. and Monoclonal Antibodies: Hybridoma and ELISA (Kodansha, (1991)) by Iwasaki, et al.

By screening an expression library using the anti-Shootinl antibody, it is possible to obtain genes that encode proteins having the activity of inducing axon formation or elongation in nerve cells. In order to prepare a cDNA expression library, cDNA molecules are inserted to λ-phage vectors, for example, and then infected into Escherichia coli. These are then spread on agar to form plaques. A nitrocellulose or nylon membrane is then placed over plaques that have reached an appropriate size (for example, incubated for eight hours at 37⁰C) to transfer the cDNA products (expressed proteins), and using a method like ordinary Western blot analysis these are subjected to analysis by the anti-Shootinl antibody to screen the cDNA clones expressing proteins having an amino acid sequence similar to that of Shootinl. For such analysis, for example, the anti-Shootinl antibody is used for the primary antibody, and the secondary antibody labeled with peroxidase or alkaline phosphatase is used to detect the primary antibody (for example, if the primary antibody is derived from rabbit, then anti-rabbit IgG antibody can be used). It is also possible to use a labeled anti-Shootinl antibody. If a candidate plaque can be obtained through this screening, then it is possible to obtain the entire cDNA clone through a known method to those skilled in the art using that cDNA fragment as a probe. Also, commercially available cDNA 5'-terminal cloning kit can be used. By introducing the clone obtained in this manner into nerve cells and assaying the axon formation and/or elongation activity, it is possible to obtain a gene for a protein that activates nerve axon formation and/or elongation.

As mentioned above, the Shootinl specific RNAi reagent can be a siRNA or a RNAi expression vector. It is not necessary that the RNAi reagent completely inhibits the expression of the Shootinl protein, and it is sufficient for it to substantially lower the amount of Shootinl protein expression within the cell. Examples

The present invention is described in detail below through examples thereof, but in no way is the present invention limited to these examples.

Example 1: Cloning Human and Rat Shootinl Gene

The present inventors recently developed a high-sensitivity two-dimensional electrophoresis method (Inagaki N. and Katsuta K, Curr. Proteomics 1, 35-39, 2004). Using this method, approximately 6,200 proteins from rat cultured hippocampal neurons were screened, detecting 277 protein spots in which expression increases in accordance with nerve axon formation.

Approximately 5,200 proteins were similarly screened, detecting 200 protein spots concentrated in nerve axons. One of the proteins detected by both of these two different screening methods was analyzed with a

MALDI-TOF/MS mass spectrometer, and as a result, ten peptides that match the amino acid sequence within the protein partially encoded by a known human cDNA (KIAA1598) were found.

Human cDNA (KIAA1598) encodes a protein having 446 amino acids, whose 5'-end has been deleted. That is, through analysis with BLAST, the inventors found that ten amino acids on the N-terminus are missing from this protein. Accordingly, we next decided to clone a gene that encodes the entire protein of 456 amino acid sequence. More specifically, with the human cDNA (KIAA1598) serving as a template, PCR was performed using the forward primer and the reverse primer having the following sequences. Forward Primer: δ'-GCGGATCCATGAACAGCTCGGACGAAGAGAAGCAGC TGCAGCTCATTACCAGT (SEQ ID NO. 10)

Reverse Primer: δ'-GCGGATCCCTACTGGGAGGCCAGTATTC (SEQ ID NO. 11)

We initially referred to the gene cloned in this manner and the protein encoded by that gene as "p52a," but finally decided to call it "Shootinl." The cDNA sequence and amino acid sequence of human Shootinl are shown in SEQ ID NOs. 1 to 3.

Using the same method as with human Shootinl, PCR was performed using the forward primer and the reverse primer having the following sequences from a rat cDNA library (Clontech) in order to clone the rat Shootinl gene. Forward Primer: δ'-CCGCTCGAGATGAACAGCTCGGACGAGGAGAAG (SEQ ID NO. 12)

Reverse Primer: δ'-CCGCTCGAGTTACTGGGAGGCCAGGATTCCCTTCAG (SEQ ID NO. 13)

The cDNA sequence and amino acid sequence of the cloned rat Shootinl are shown in SEQ ID NOs. 4 to 6.

Example 2: Analysis of Spatial and Temporal Expression of Shootinl in Rat Preparation of Anti-Shootinl Antibody

The GST-fused protein of human Shootinl cloned through the above method was expressed using E. coli and purified using a Glutathion Sepharose 4B column. GST was then removed by cleaving it from the purified protein using protease, and the Shootinl that was obtained was immunized to rabbit to produce antibodies according to routine procedure. The antibodies that were obtained were column-purified with Shootinl as a ligand, and the purified antibodies were used in the following experiments. Immunoblotting

Samples prepared from various tissues of Wistar rats were treated with SDS and 15 μg of each sample was separated by 10% polyacrylamide gel. The proteins were then transferred to a PVDF film and Shootinl was detected using the above anti-Shootinl antibodies (100Ox dilution), HRP-labeled anti-rabbit IgG antibodies (2000χ dilution), and ECL reagents (Amersham Biosciences). The result was that, as shown in FIG. 3, Shootinl was expressed specifically in the brain, and there was a large rise in the amount of expression the fourth day after birth (P4), during which axons are formed.

Example 3: Analysis of Cellular Distribution of Shootinl in Rat Cultured

Hippocampal Neurons

Preparation of Rat Cultured Hippocampal Neurons

The hippocampus of a Wistar rat embryo 18 days (E 18) was isolated and digested by papain enzyme to prepare segregated nerve cells. The nerve cells obtained in this manner were cultured at 37⁰C and 5% CO2 in a culture medium (Neurobasal Medium, B-27 supplement, 1 mM glutamine, 2.5 μM cytosine β-D-arabinofuranoside) on cover glass coated with poly-D-lysine and laminin.

Immunostaining Rat Cultured Hippocampal Neurons

Rat cultured hippocampal neurons on the third day after cultured were fixed on ice for ten minutes with 3.7% formalin and then subjected to membrane permeabilization for ten minutes with -2O⁰C methanol. The cells were then incubated at 4⁰C for one day with the anti-Shootinl antibody

(5000χ diluted) as the primary antibody and then incubated for one hour at room temperature with ALEXA488-labeled anti-rabbit IgG antibody (100Ox diluted) as the secondary antibody to visualize Shootinl. The result is shown in FIG. 4. As shown in the drawing, a high concentration of Shootinl was confirmed at the growth cone of an elongating axon tip in the cultured nerve cells prepared from the hippocampus of a rat embryo 18 days (E 18).

Example 4: Analysis of Nerve Axon Formation Induced by Exogenous Expression of Shootinl

Human Shootinl with Myc-tag was subcloned to the expression vector pCAGGS for mammalian cell, having an actin promoter (see Niwa et al., Gene 108 (1991), pl93-200) to produce a Shootinl expression vector.

This vector was introduced into the nerve cells prepared from the hippocampus of a Wistar rat 18 days (E 18) using Nucleofector™ (AMAXA Biosystems), and the cells were then cultured on cover glass. On the seventh day in vitro, the nerve cells were fixed as described above, and using anti-myc antibodies, exogenous Shootinl were visualized. Further, change of the nerve cells expressing exogenous Shootinl was analyzed. The result is shown in FIG. 5. As shown by the arrow in this drawing, overexpression of exogenous Shootinl in cultured hippocampal neurons induced the formation of a plurality of axons.

FIG. 6 shows cell localization of Shootinl when Shootinl is expressed exogenously in cultured rat hippocampal neurons. More specifically, the hippocampus was isolated from a rat embryo 18 days (E 18) and subjected to dispersed culture for two days. The cultured nerve cells were then transfected with a human Shootinl (with a myc-tag) expression vector, and then after five days Shootinl in the nerve cells was detected using immuno staining (primary antibody: anti-myc x400, secondary antibody anti-rabbit χ800). The upper panel of FIG. 6 is a complete view of the nerve cells, and the lower panel is a magnified view of the area around the axonal growth cone, surrounded by a square of the upper panel. As shown in FIG. 6, Shootinl was found at a high concentration in the growth cone at the axon's tip even when Shootinl was expressed exogenously.

It was also observed through analysis by a time lapse microscope that Shootinl moves dynamically in the nerve growth cone.

Industrial Applicability

As discussed above, the present invention can be utilized in the development of a novel nerve regeneration technology. For example, it can be employed in research and establishment of axon regeneration medical technologies, such as an effective remedy for damages in central and peripheral nerves caused by stroke or spinal cord injury. More concretely, the present invention has various industrial applicability including development of new medicines and clinical application, such as use in new medical treatments to stimulate or promote the axon regeneration by expressing Shootinl, having an axon formation or elongation activity, in the damaged nerve cells of patients via a vector such as a virus, and use in development of axon regeneration therapeutics by using Shootinl as a probe to screen compounds that activate Shootinl.

Claims

1. A method for inducing formation or elongation of axons in nerve cells, comprising: positively controlling an expression or activity of nerve growth cone localized molecule Shootinl or a splicing variant thereof.

2. The nerve axon formation/elongation inducing method according to claim 1, wherein expression of Shootinl or its splicing variant within nerve cells is positively controlled by introducing a gene for Shootinl or its splicing variant into those cells.

3. A nerve axon formation/elongation inducing agent, including a recombinant expression vector constructed such that a gene for nerve growth cone localized molecule Shootinl or a splicing variant thereof is expressed in nerve cells.

4. The nerve axon formation/elongation inducing agent according to claim 3, wherein the recombinant expression vector is constructed so as to express a gene for human-, rat-, or mouse-derived Shootinl or a .splicing variant thereof.

5. A gene therapeutic agent for nerve regeneration, including a recombinant expression vector constructed such that a gene for nerve growth cone localized molecule Shootinl or a splicing variant thereof is expressed in nerve cells.

6. The gene therapeutic agent for nerve regeneration according to claim 5, wherein the recombinant expression vector is constructed so as to express a gene for human-, rat-, or mouse-derived Shootinl or a splicing variant thereof.

7. A human-derived Shootinl protein indicated by (a) or (b) below:

(a) a protein having the amino acid sequence of SEQ ID NO. 3;

(b) a protein having an amino acid sequence obtained by deletion, replacement, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO. 3, and having an activity of inducing axon formation or elongation in nerve cells.

8. A rat-derived Shootinl protein indicated by (a) or (b) below:

(a) a protein having the amino acid sequence of SEQ ID NO. 6;

(b) a protein having an amino acid sequence obtained by deletion, replacement, or addition of one or several amino acids in the amino acid sequence of SEQ ID NO. 6, and having an activity of inducing axon formation or elongation in nerve cells.

9. A Shootinl gene, encoding any one protein set forth in claim 7 or 8.

10. A DNA indicated by (a), (b) or (c) below: (a) a DNA having the base sequence of SEQ ID NO. 1 or 4;

(b) a DNA that has a base sequence hybridized under stringent conditions to DNA having a base sequence that is complementary to the base sequence of SEQ ID NO. 1 or 4, and that encodes a protein having an activity of inducing axon formation or elongation in nerve cells;

(c) a DNA that is obtained by screening an expression library using an anti-Shootinl antibody and that encodes a protein having an activity of inducing axon formation or elongation in nerve cells.

11. A method for screening a therapeutic agent for nerve regeneration, comprising: screening a compound that positively controls an expression or activity of nerve growth cone localized molecule Shootinl or a splicing variant thereof.

12. An antibody, for research relating to nerve axon formation or elongation or to nerve regeneration, wherein the antibody specifically detects Shootinl or a splicing variant thereof.

13. The antibody according to claim 12, wherein the antibody is anti-Shootinl antibody, which detects human- or rat-derived Shootinl protein specifically.

14. The DNA indicated by (c) of claim 10, wherein the DNA is obtained by screening an expression library using the antibody of claim 12 or 13.

15. An RNAi reagent, for research relating to nerve axon formation or elongation or to nerve regeneration, wherein the RNAi reagent is introduced into cells so as to suppress specifically an expression of Shootinl or a splicing variant thereof.