JP2003512300A - Axonal outgrowth and induction by tenascin-C - Google Patents

Abstract

  (57) [Summary] We have developed a test method to quantify the neurite response at the sensitive support interface, and when poly-L-lysine or fnA-D is given as an interfacial option, the neurites become fnA It has been found that it shows a strong priority for -D. In addition, axons were found to favor cells that overexpress the largest, but not the least, tenascin-C splice variant over the choice of control cells and cells transfected with tenascin-C. . The permissive guidance cue for large tenascin-C expressed by cells was mapped to fnA-D, indicating that neurite outgrowth and induction is promoted by distinct sequences within fnA-D. Thus, neurite outgrowth and neurite outgrowth, regulated by alternatively inherited regions of tenascin-C, are discrete events that can be independently regulated.

Description

Description: BACKGROUND OF THE INVENTION G. FIG. NIH R01NS24168 and S.I. M. N to
Supported by the IEHS Exploratory Research Award RQ 1610. This application is related to US Provisional Application No. 60 / 132,13, filed May 1, 2000.
Includes material No. 7 and cites its priority. BACKGROUND OF THE INVENTION Tenascin-C has been implicated in regulating both neurite outgrowth and induction. We have previously shown in testers that certain areas of tenascin-C have potent neurite outgrowth-promoting activity. This region consists of alternating fibronectin type 3 (FN-3) repeats AD, abbreviated fnA-D
Is described. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the multi-domain structure of human tenascin-C. FIG. 2 is a schematic diagram showing an axon guidance assay. FIG. 3A shows that alternatively inherited regions of tenascin-C provide a permissive axon guidance guidance cue. FIG. 3B shows that alternatively inherited regions of tenascin-C provide a permissive axon guidance guidance cue. FIG. 4A shows that fnA-D overcomes the tenascin-C boundary and axons progress. FIG. 4B shows that fnA-D overcomes the tenascin-C boundary and axons progress. FIG. 5 shows that neurite outgrowth promoting sites in fnD do not regulate neurite outgrowth. FIG. 6A shows that axon guidance is locally restricted to the C-end of fnA-D. FIG. 6B shows that axon guidance is locally restricted to the C-end of fnA-D. FIG. 7A shows that fnC is involved in regulating axon guidance. FIG. 7B shows that fnC is involved in regulating axon guidance. FIG. 8A shows that fnA-D induces neurites within cellular tenascin-C. FIG. 8B shows that fnA-D induces neurites within cellular tenascin-C. FIG. 9A shows neurite outgrowth in the presence of Tenascin-C antibody. FIG. 9B shows neurite guidance in the presence of tenascin-C antibody.

[Procedure amendment] [Date of submission] December 27, 2001 (2001.12.27) [Procedure amendment 1] [Document name to be amended] Description [Item name to be amended] Full text [Amendment method] Change [Amendment] Title: Extension and induction of neurites by tenascin-C. Claims 1. An eight amino acid sequence V defined by a one-letter amino acid code
A peptide consisting of FDNFVLK, comprising no more than 75 amino acids.
Puchido. 2. The method according to claim 1, wherein the peptide consists of no more than 50 amino acids.
2. The peptide according to 1. 3. The method according to claim 2, wherein the peptide consists of no more than 20 amino acids.
2. The peptide according to 1. 4. The method according to claim 1, wherein the peptide consists of no more than 10 amino acids.
2. The peptide according to 1. 5. The amino acid according to claim 5, wherein the peptide is not linked to another amino acid.
2. The peptide according to claim 1, which is of the sequence VFDNFVLK. 6. Growth and / or induction of axons and / or dendrites
A method for stimulating the administration of a peptide according to claim 1 to a neuron.
The method that consists of doing. 7. The method of claim 6, wherein said neurons are in a human. 8. The method of claim 7, wherein said peptide is provided to the spinal cord. 9. The method of claim 8, wherein said peptide is provided by injection.
Method. 10. Growth and / or induction of axons and / or dendrites
A method for stimulating conduction, comprising administering a vector to a damaged nervous system.
This vector comprises a nucleotide sequence coding for the peptide of claim 1.
A nucleic acid comprising a nucleic acid. 11. The method of claim 11, wherein said nucleic acid molecule is present in a virus at the time of administration.
Item 10. The method according to Item 10. 12. The growth and / or induction of axons and / or dendrites.
A method for stimulating conduction, which involves administering a peptide to a neuron.
The peptide is at least 7 amino acids in length;
The tenascin-C region consists of all or a part of the C region.
, FnD and fnC. 13. The method according to claim 13, wherein the peptide is an eight amino acid peptide VFDNFVLK.
13. The method according to claim 12, consisting of: 14. The method according to claim 14, wherein the peptide is outside the tenascin-C region and
Claims that does not include any of the tenascin-C amino acid sequences that exceed amino acids.
14. The method according to 13. 15. The peptide according to claim 10, wherein the peptide is identical in length to the tenascin-C region.
The same amino acid sequence and the tenascin
The C region is aligned and numbered sequentially from the same end, these two arrangements
When the like-numbered amino acids in a column are compared, the amino acids in the same sequence are
At least N percent identity to the amino acids of the tenascin-C sequence
13. The method of claim 12, wherein N is 70. 16. The method of claim 15, wherein N is 80. 17. The method of claim 16, wherein N is 90. 18. The method of claim 12, wherein said neurons are in a human.
Law. 19. The method according to claim 19, wherein the neuron is human and the peptide is spinal cord.
13. The method of claim 12, wherein the method is provided. 20. The administration of the peptide causes the injured nervous system to
A nucleic acid vector comprising a region code for
Achieved by infection with a virus containing nucleic acid consisting of
13. The method according to claim 12. 21. The method of claim 12, wherein the tenascin-C region is fnA-D.
The described method. 22. The tenascin-C region is fnD.
the method of. 23. The tenascin-C region of claim 12, wherein the tenascin-C region is fnC.
the method of. 24. The object stimulates axonal and / or dendritic growth.
13. The method of claim 12, wherein 25. The method according to claim 25, wherein the object is independent of axonal guidance,
25. The method of claim 24, wherein the method is to stimulate dendritic growth. 26. The method according to claim 26, wherein the object stimulates the induction of axons and / or dendrites.
13. The method of claim 12, wherein 27. The method according to claim 19, wherein the objective is to grow axons and / or dendrites.
And stimulating the induction of axons and / or dendrites.
7. The method according to 6. 28. fnA-D, fnD, fnC, and fnA-D, fnD
And a selected peptide region selected from the group consisting of homologues to fnC
A peptide that does not include the tenascin-C region that is not in this selected peptide region,
Exceeds L amino acids in length, and homologs of the region are
The same, if the area is aligned end-to-end, the area is at least N
Percent identical amino acids but 100% identical amino acids
Wherein N is 70 and L is 100.
. 29. The peptide according to claim 28, wherein L is 10. 30. Growth and / or induction of axons and / or dendrites
29. A method for stimulating conduction, comprising applying the peptide of claim 28 to a neuron.
A method comprising administering. 31. The method according to claim 31, wherein the peptide is administered to an integrin receptor prior to administration of the peptide.
Is added to the neuron via a vector or virus.
Thereby increasing the number of integrin receptors in said neuron;
A method according to claim 6, 12 or 28. 32. Response of neurons to factors that promote growth or induction
Neurites are used to increase the number of integrin receptors
Altering the DNA content of the axon to include the axon. Description: BACKGROUND OF THE INVENTION G. FIG. NIH R01NS24168 and S.I. M. N to
Supported by IEHS Exploratory Research Award RQ 1610
It was held. This application is related to US Provisional Application No. 60 / 132,13, filed May 1, 2000.
Includes material No. 7 and cites its priority. BACKGROUND OF THE INVENTION Tenascin-C has been implicated in regulating both neurite outgrowth and induction.
I have. We have previously identified that certain areas of Tenascin-C
Neurite outgrowth promoting effect. This area is alternatively inherited
Consists of fibronectin type 3 (FN-3) repeats AD, abbreviated fn
A-D. The development of the nervous system is absolutely dependent on the growth of targeted axons and dendrites
You. Not only do neuronal processes extend to reach the correct goal (axons
Process extension) and must pass in the right direction (axon guidance). Axon
Elongation (Smith et al., 1986) and induction of neurites (Letourneau et al., 199).
Both are thought to be regulated by astrocyte-derived surface molecules.
Some of these molecules temporarily appear at the boundaries of migration pathways within the developing cortex (Stei
ndler et al., 1989), regenerating glial scarring in the adult central nervous system (CNS).
(McKeon et al., 1991: Laywell et al., 1996; Lochter et al., 199
1) There is an extracellular matrix protein, tenascin-C. From that position,
Neissin C naturally prevents elongation of neuronal processes and / or
It was thought to be a barrier to diversion (Steindler et al., 1989). And
However, in vivo (Gates et al., 1996; Gotz et al., 1997; Zhang et al.,
1997) and within the examiner (Faissner and Kruse, 1990: Lochter et al., 1991 'Mei
ners and Geller, 1997), showed that tenascin-C
To provide an acceptable and also inhibitory cue for the growth of
It became clear that we could do it. [0005] Tenascin-C is an alternative, rather than a single molecule, with potentially diverse actions.
(Chung et al., 1996; Gotz et al., 199).
7; Meiners and Geller, 1997). Tenascin-C splice variant
Only the number of FN-3 domains differs, for example,
Large splice variants are included in the smallest splice variants
Have seven alternatively inherited FN-3 domains (fnA-D in FIG. 1)
With a sign). Increased cell migration and stages of axonal growth in the developing CNS
Is closely related to the expression of large tenascin-C, not small tenascin-C.
(Crossin et al., 1989; Steindler et al. 1989; Kaplony et al., 1991; Ba
rtsch et al., 1992). And, this means that fnA-D
It suggests that it may promote neurite outgrowth. Our own, Tennessee
By structure-function studies of isin C splice variants, fnA-D
As a sexually expressed protein by itself and as part of a large tenascin-C,
Within the examiner, it can strongly promote neurite outgrowth from various types of neurons
I understand (Meiners and Geller, 1997: Meiners et al., 1999). SUMMARY OF THE INVENTION An object of the present invention is to stimulate the growth and induction of axons and / or dendrites.
Is to develop a method. Other objects and advantages of the invention will be set forth in the following description of the invention.
It will be apparent to those familiar with the technology. [0007] In one general aspect, the invention is defined by a one-letter amino acid code.
A peptide consisting of the eight amino acid sequence VFDNFVLK. This peptide
In certain embodiments, where it is appropriate to use smaller peptides, more than 75
Consists of no amino acids. Peptides have no more than 50 amino acids, no more than 20
More preferably, it is an amino acid, more preferably not more than 10. other
An eight amino acid peptide that is not linked to an amino acid is an invention by itself. In one related aspect, the invention comprises fnA-D, fnD, and fnC.
It is a peptide consisting of the tenascin-C region selected from the group. This peptide
Indicates that other than ten amino acids in length, other than the selected tenascin-C region,
Does not include the isin C region. (This peptide is a tenascin other than the selected tenascin-C region.
More preferably, it does not include the isin C region and exceeds 10 amino acids in length.
). [0009] In another aspect, the invention relates to the growth and / or growth of axons or dendrites.
Is a method for stimulating induction. This method uses the pen described in the previous two paragraphs.
Consists of administering the peptide to the neuron. In one embodiment, this
These axons or dendrites are present in the human nervous system. For example, the peptide
Supplies the spinal cord. In one delivery mode, the peptide is delivered by injection.
In a related aspect of the invention, the peptide
A vector, which is a nucleic acid comprising a base sequence code, is administered. Alternative approach
Thus, upon administration, the nucleic acid molecule is within the virus. In a related aspect, the invention involves stimulating axonal or dendritic growth or induction.
It is a way to intensify. This method can be applied to axons or dendrites in length
Administering a peptide that is at least 7 amino acids. This peptide
, FnA-D, tenascin-C region selected from the group consisting of fnD and fnC
Consists of all or part of the territory. In certain embodiments of this aspect,
Tide consists of the eight amino acid peptide VFDNFVLK. For example, the peptide
Tenascin-C amino acids outside the tenascin-C region and over 100 amino acids
Does not contain any of the arrays. Further, in one aspect related to this method,
The peptide is a homologous peptide sequence identical in length to the tenascin C region
Consists of Therefore, the amino acid sequence of the same type and the tenascin C region are aligned,
Numbered consecutively from the same end, the same numbered array of these two sequences
When amino acids are compared, amino acids of the same sequence and amino acids of the tenascin-C sequence
Are at least N percent identical. In this case, even if N is 70,
Good, but it is more preferable that N is 80, and even more preferable that N is 90.
No. To illustrate the calculations, the following two aligned virtual 20-mer peptides are
Consider. [0011] AAAAAAAAAAVVVVVVVVVV AAAAAAAAGGGGGGGVVVVVVV There are homology at 14 of the 20 positions along the sequence. Therefore, N is 70
is there. In one general aspect, the object of the method of the invention is to provide axons and / or dendrites
To stimulate the growth of the projections. In particular embodiments, axons and / or
Or to promote axonal growth independently of the induction of dendrites. vice versa,
In another general aspect, the purpose is to induce axons and / or dendrites
It is to stimulate. And in particular embodiments, axons and / or trees
Stimulate the induction of axons and / or dendrites independently of dendritic growth
And [0013] Any of the methods of the invention can use any of the peptides of the invention. Also
In another aspect, the present invention provides a DNA molecule code for a peptide of the present invention.
You. A preferred embodiment for all peptides of the invention is the purified peptide, ie
For example, a purified preparation of the peptide, such as an isolated peptide. Synthetic peptides, such as those synthesized in non-human cells, especially non-mammalian cells, are
This is a preferred embodiment. Similarly, a preferred embodiment for all DNA molecules of the invention is a purified DNA molecule.
Ie, a purified formulation of the DNA molecule, eg, an isolated DNA molecule
It is. Such preferred embodiments are made by recombinant DNA technology, or
Synthetic DNA molecules that have been replicated and synthesized in non-human cells, especially non-mammalian cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples and examples are intended to illustrate the present invention and are not intended to be limiting.
It is not specified. [Abbreviations] The following abbreviations are used in this application. CFDA, carboxy fluorescein
-Diacetate. CNS, central nervous system. CSPG, chondroitin sulfate proteo
Glycan. EGF, epidermal growth factor. fbg, fibrinogen. FN-3, Five
Lonectin type 3. fn, FN-3 domain. mAb, monoclonal antibody.
pAb, polyclonal antibody. TN. L, large tenascin splice barrier
And TN. S, small tenascin splice variant. The following one-letter code is used to represent an amino acid. S. Serine, T. Sure
Onin, N-asparagine, Q-glutamine, K-lysine, R-arginine, H
-Histidine, E-glutamic acid, D-aspartic acid, C-cystine, G-g
Lysine, P-proline, A-alanine, I-isoleucine, L-leucine, M-
Methionine, F-phenylalanine, W-tryptophan, V-valine, Y-thio
Rosin, X-any amino acid. Further, the following one-letter code is used to represent a nucleic acid. A-adenine, C-si
Tosin, G-guanine, T-thymidine, R represents A or G, Y represents T or
Represents C, and N represents any nucleic acid. [Amino acid sequence of tenascin-C and its DNA code] The following amino acid and nucleic acid sequences are listed in Genbank catalog number X78565 version.
X785565, the human tenascin C sequence from
For locus HSTENAS3, 7560bp mRNA, and tenascin-C
Human mRNA, 7560 bp. [Table 1] PolyA signal 7522 ... 7527 [Small Region of Tenascin-C] The peptide VFDNFVLK is composed of amino acids 1646-1653 (SEQ ID NO: 1).
). FnA-D is amino acids 1072-1078 (SEQ ID NO: 8). The alternatively inherited FN-3 domain D, fnA-D, contains amino acids 1618
-1708 (sequence identification number 9). The alternatively inherited domain C, fnA-D, comprises amino acids 1527-161
7 (sequence identification number 10). FnD is another name for the alternatively inherited FN-3 domain D. FnC is another name for alternatively inherited FN-3 domain C. [Integrin alpha 7 precursor sequence] This amino acid sequence, related information and abstract are described in NCBI, Entrez Protein
Obtained from the database. Locus NP_002197 1137aa PRI October 1, 1999
Concept Definition Integrin alpha 7 precursor [Homo sapiens]. Inventory NP — 002197 Version NP — 0021977.1 GI: 4504753 ITGA7 encodes integrin α chain 7. Integrins are alpha chain and
It is a heterodimeric integral membrane protein composed of β chains. α chain 7 is extracellular domain
In vivo, post-translational fission occurs and light and disulfide-linked disulfides bind to β1.
An inn that forms heavy chains and binds to the extracellular matrix protein laminin-1
Forming tegulin. [Table 3] This nucleic acid sequence, related information and summaries are provided by NCBI, Entrez Nucleotide
Obtained from the database. Locus NM_002206 4079 bp mRNA PRI 1999
October 1 Concept Regulation Homo sapiens integrin alpha 7 (ITGA7) mR
NA. Catalog NM_002206 Version NM_002206.1 GI: 4504752 [Integrin Beta 1 Sequence] The amino acid sequence, related information and abstract are available at NCBI, Entrez Nucleotide
Obtained from the database. Locus HSFRNB 3614 bp mRNA PRI April 1, 1999
2 Day Concept Definition Human mRNA for integrin beta 1 subunit. Catalog X077971 Version X07979.1 GI: 31441 [Table 6] EXAMPLES Example 1 The purpose of this study was to determine whether fnA-D also provides an axonal guidance cue.
Investigate whether the same or different sequences regulate elongation and induction.
And We want to quantify neurite responses at distinct substrate boundaries
Method was developed and options were given at the poly-L-lysine / fnA-D interface.
In this case, it was found that neurites displayed a strong preference for fnA-D. further,
If there is a choice between control cells and cells transfected with tenascin-C, neurites
, Not the smallest Tenascin-C splice variant, but the largest Tenascin-C
It was found that cells overexpressing the syn-C splice variant were preferred.
Mapping the permissive induction cue of large tenascin-C expressed by cells to fnA-D
And a bacterially expressed protein corresponding to a particular alternatively inherited FN-3 domain
And a monoclonal antibody against the neurite outgrowth promoting site
It is clear that process extension and induction are facilitated by distinct sequences within fnA-D.
I made it clear. Therefore, it is adjusted by the alternatively inherited area of Tenascin-C.
Neurite outgrowth and neurite outgrowth are discrete events that are independently regulated
. In previous studies, we conclude that neurite outgrowth and induction may be separable events.
(Powell and Geller, 1999). For this reason, fnA-D
Two complementary alternatives explore the hypothesis of giving distinct elongation and guidance cues
An analysis method was used. In one assay, the growing axons were selected to be poly-L-lysine.
Uniformly inherited universal tenascin CFN-3 domain, also large and small
Purified expression protein corresponding to the tenascin-C splice variant
You can choose. The other assay describes the effect of fnA-D in cell tenascin-C.
It was designed to more closely resemble the in-vivo environment in order to investigate. axis
Chordal processes overexpress large or small tenascin-C in heterogeneous monolayers
To allow selection between transfected and untransfected cells. Size on inert substrate
Axons that were obscured by tenascin-C were strong against fnA-D
A preference was only revealed for large tenascin-C on the cell substrate (Me
iners and Geller, 1997; Meiners et al., 1999). To the neurite outgrowth promotion site
Corresponding to a specific alternatively inherited FN-3 domain
Induction and extension promotion using expressed proteins to different sequences in fnA-D
And further determined the position. Thus, neurite outgrowth and induction is not affected by tenascin
Individual adjustments can be made via alternatively connected regions of C. Materials and Methods Proteins and Antibodies Transfected Baby Hamster Kidney (BHK) Cells, Bacterial Expression Protein
Quality and rabbit polyclonal tenascin-C antibodies were obtained from Harold Erickson
Ph.D. (Cell Biology, Duke University Medical Center, Durham, NC)
Of the human tenascin-C in the transfected cells.
Liant was produced (Aukhil et al., 1993). Gelatin Sepharose and HI
The culture supernatant of these cells can be analyzed by droxyapatite chromatography.
Purify natural large and small tenascin-C (Aukhil et al., 1990;
Erickson and Briscoe, 1995), followed by electroelution from non-denaturing gels (Ho, S
-Y., Palnitkar, S., and Meiners, S., unpublished data). Bacterial expressed protein
(Aukhil et al., 1993) describe the universal FN-3 domains 1-5 and 6-8 (f
n1-5 and fn6-8), an alternatively succeeded FN-3 domain of large tenascin-C
From the main fnA-D, and the alternatively inherited domain, the FN-3 domain
This corresponds to fnA-D (-) C from which Cn (fnC) was subtracted. fn1-5, fn
6-8 and fnA-D are used as a polymerase chain reaction (PCR) and as a template.
Generated using cDNA isolated from BHK cells transfected with large tenascin-C
did. PCR and isolated from U251-MG glioma cells as template
fnA-D (-) C was generated using the cDNA. (The species with fnA-D
Although predominant, U251-MG cells express fnA-D and fnA-D (-) C.
Alternatively, a transcript of tenascin-C is generated. (Erickson and Bou
rdon, 1989)) from U251 MG cells, which are almost completely large tenascin-C.
Rabbits against highly purified tenascin-C (Erickson and Bourdon, 1989)
A polyclonal, full-length tenascin-C antibody is prepared and the corresponding expression vector is prepared.
Rabbit polyclonal against fn1-5 and fnA-D for proteins
The antibody was prepared. All reagents mentioned correspond to human proteins. The N-terminal region fnA1-A4 of fnA-D and the C-terminal region fn of fnA-D
Bacterial expressed proteins corresponding to BD are Harold-Ericsson and Franco
Dr. Franscoise Coussen et al. (Bold, Bordeaux, France)
ー University) gift. Both correspond to the human protein. The monoclonal antibody J1 / tn2 against murine tenascin-C was Andrea
Dr. Andreas Faissner (D-69, Heidelberg, Germany)
120, Department of Neurobiology, Heidelberg University). J1 / tn2
Is contained on the fnD of mouse tenascin-C (Gotz et al.
al., 1996). Chicken (mainly composed of neurocan, phosphacan, versican and aggrecan)
The CSPG mixture isolated from embryonic brain was purchased from Chemicon International
(Temecula, Ornia). Agreca (St. Louis, MO)
Was obtained from Sigma Chemical Co, and laminin-1 was obtained from Sigma Chemical Co.
Obtained from GIBCO BRL (Rockville, MD). Natural chondro
Monochrome that reacts with glycosaminoglycan moiety of itine sulfate proteoglycan
Antibody CS-56 was obtained from Sigma Chemical Co., Ltd.
Antibody RT97 (Iowa City, Iowa) is a developmental research hybridoma
From the Developmental Studies Hybridoma Bank and from the neurofibrils 20
Polyclonal antibodies against 0 were obtained from Sigma Chemical Company. In addition, human
Monoclonal antibody tenascin-recognizing an antigenic determinant in fnB of tenascin-C
3B was obtained from Chemicon. [Culture of neuron cells] Culture of cerebellar granule neurons was prepared based on the description of Levi et al. (1984).
Was. 5 ml of B containing 2M, HEPES buffer for culturing neurons
Eight days after birth (P8) into a petri dish containing ME (BME-HEPES)
The brain was collected from them. Remove the cerebellum and remove the meninges and blood vessels from the endothelial cells
To ensure minimal contamination. Dissect the cerebellum with a dissecting knife (<0.5m
m) and then put in BME-HEPES containing 0.025% trypsin
And incubated at 37 ° C. for 10 minutes. After incubation at 0.025% soybean trypsin
By adding 1 ml of BME containing 0.05% of DNase I
Stop the trypsinization and then heat-paste the Pasteur pipette
, The tissue was crushed softly and dispersed in a homogeneous suspension. This suspension is
Transfer to a fresh tube and then to residual tissue mass, DMEM-25 mM, KCl / 10%
Heat-inactivated FC (3-4 ml) was added and grinding was repeated. Ethanol sterilized
Filter the cells through a 40 μm mesh and centrifuge at 1500 rpm for 10 minutes.
Heart-separated. The spheres of the cerebellar granule neurons were treated with DMEM-25 mM, K
Resuspend in Cl / 10% FCS and induce neurite outgrowth and neurite outgrowth as described below
Used for analysis. [Axonal guidance analysis method] Axonal guidance is defined as guiding neurite movement with considerable certainty.
I do. The two most frequently used induction assays are stripe analysis (Vi
elmetter et al., 1990) and spot analysis (Snow et al., 1991). And
However, none of the assays quantify the neurite response. There
So, we have PLL and tenascin CFN-3 expressed protein, natural tenas
Axons at the interface formed with Thin C splice variant or CSPG
The spot analysis was modified to quantify the protrusion response. 5 μl drop of target
(300 nM in Hanks buffered saline, HBS)
The PLL-protein interface is located at the center of the 2 mm PLL-coated cover glass.
Generated. Cover glass with protein drops in a 24-well tray.
After incubation at 7 ° C. for 2 hours, excess protein solution was washed away with HBS. NHS
Tan joined to Luoresen (Pierce Chemical, Rockford, Illinois)
By incubating all cover glass with parkin, tenascin-C splice
A similar coating efficiency (approximately 5 pmol / cm) ^Two A) demonstrated that there is. After 2 hours, coat by adding 2% SDS
Protein was removed. Then, we are involved in the protein associated with the cell monolayer.
As described earlier (Meiners et al., 1999)
Fluorescence of the protein was compared to the cytofluor II (Cytofluor II) fluorescence microplate.
Card Reader (Perceptive Biosystems, Framingham, MA)
(PerSeptive Biosystems). Other findings (Dorrics et al.,
1996: Fischer et al., 1997), observe large differences in coating efficiency
I couldn't do that. Cerebellar granules at a concentration of 60,000 neurons / well on cover slips
And culture in DMEM-25 mM, KCl / 10% FCS for 48 hours
did. This time, the cover glass was treated with acetic acid / ethanol (5% / 95%) for -20.
Fix for 5 minutes at ° C. After fixing, the cover glass was washed with PBS (pH 7.4).
0.14 M NaCl, 2.7 mM KCl, 1.5 mM KH ₂ PO ₄ , 4.3
mM NaHPO ₄ ), PBS containing 10% FCS (PBS-serum)
A suitable, first antibody against a protein in a 1: 100 diluted drop in
Polyclonality for nasin C splice variants and expressed proteins
Full length Tenascin-C antibody or monoclonal antibody to CSPG
CS-56). After washing in PBS, the cover glass is
Incubation with fluorescein-conjugated, second antibody diluted 1: 100 in S-serum
(Goat anti-rabbit secondary antibody to Tenascin-C spot, and CSP
Goat anti-mouse secondary antibody to G spot) (Durham, NC)
Organon Technica Kappel). Wash again with PBS and remove Tenascin-C spot.
The cover glass containing the immunoglobulin is used together with the monoclonal antibody RT97 against neurofibrillary tangles.
And then incubated with a rhodamine-conjugated, goat anti-mouse secondary antibody. The other
, Those containing CSPG spots are polyclonal anti-
Incubated with body, then with rhodamine-conjugated, goat anti-rabbit secondary antibody
. All first and second antibody incubations were performed at 4 ° C. for 30 minutes. Cover gala
In PBS and then ddH ₂ Wash in O 2, then
Fluoromount-G on Birmingham, Birmingham, Alabama
Zan Biotechnology Associate). In parallel culture
Nonspecific binding of the second antibody was controlled by removing the appropriate first antibody.
. Zeiss Axiop with extra-fluorescent illuminator with appropriate set of filters
Fluorescent dyes were visualized using a run (Zeiss Axioplan) microscope and the cultures were examined.
For capture of culture images, use the Macintosh Quadra 700 with Sion
For use with LG-3 frame grabber board
Was. Remains on the substrate (by stopping at the interface or turning)
Or the number of neurites on each side of the PLL / protein interface on the other side
The images were analyzed by counting. For both sides of the interface for each condition
, 75 axons were considered as samples. Figure 2 shows our modified neurite outgrowth.
It is the schematic which shows a derivative analysis method. In the analysis, 10 μm from the protein / PLL interface
Of these, only single non-bundle neurites were considered. This distance is 10-5
It was selected from the fact that it extends 0 μm (Gomez and Letourneau, 1994). further
Count only the axons moving toward the interface (the angle between the axons and the interface is 90 °
Axons with cell bodies lying on the interface were not counted. It
From the PLL to the target protein or from the protein to the PLL
The percentage of neurites was assessed. [Axonal outgrowth analysis method] In order to investigate the neurite outgrowth promoting properties of fnA-D versus fnA-D (-) C,
The PLL-coated cover glass in the 24-well tray was washed with the expressed protein (HBS
(250 nM in) at 37 ° C. for 2 hours. In some experiments, the cover
The glass was treated with a mixture of fnA-D and monoclonal antibody J1 / tn2 (75 μg /
ml). Rinse excess protein solution with HBS and remove cerebellar granules
Neurons are placed on coverslips at a concentration of 60,000 neurons / well,
Axons grow in DMEM-25mM, KCl / 10% FCS for 48 hours
Caught. Subsequently, the extent of neurite outgrowth was adjusted to carboxyfluorescein
Judgment was made by tart (CFDA) labeling (Petroski and Geller, 1994). C
The FDA (Sigma Chemical Co.) has been developing
Kiki is strongly dyed. The image of the culture was transferred to the Macintosh Quadra 70
0 at NIH Image Software (http://rsb.info.nih.gov/
(Available). Protrusion equal to or greater than one cell body
As a sample, 100 neurons with origins were considered for each condition. Each first
The total length of the process and its branches was measured for each neuron, and the length of each individual axon was measured.
The total length of the axons was calculated as the sum of the lengths. [Axonal Induction Assay on Cell Substrate] To examine the regulation of neurite outgrowth within a cell, we used Powell's
Non-transfection expressing no Tenascin-C according to their modified method (1977).
BHK cells and the largest or smallest tenascin-C splice base
Cells with transfected BHK cells that overexpress Liant (Aukhil et al., 1993).
A cell interface was created. First, transfected BHK cells were prepared according to the manufacturer's instructions.
Was labeled with a red fluorescent cell linker PKH26 (Sigma Chemical Co., Ltd.) according to
. This dye performs selective sorting without transferring the label to unlabeled cells.
Irreversibly in the cell membrane (Ford et al., 1996).
A single cell suspension of transfected and non-transfected cells was then added at a 1:10 ratio.
And mix the cell mixture with 1 × 10 ^Five Cell concentration
And applied to the PLL coated cover glass in a 24-well tray. At this concentration
Shows a confluent monolayer after 24 hours, transfection labeled with individual PKH26
Cells were readily identifiable as "islets" interspersed between non-transfected cells. form
Transfected cells can also be transformed from untransfected cells by the immunoreactivity of tenascin-C.
It was easily identifiable. Cerebellar granule neurons were cultured in BMEM in DMEM-25 mM, KCl / 10% FCS.
It was applied on the K monolayer and the axons were allowed to stretch for 48 hours. This time, neuro
And their protrusions were labeled with CFDA. Capture culture images
Analysis of neurite responses on both sides of the interface formed between transfected and transfected cells
Was. Produced on non-transfected cells and remained or transfected on non-transfected cells
Assess the number of neurites that have spread to the cells, and likewise arise on transfected cells and transfect
The number of axons that remained on the cells or spread to untransfected cells was assessed.
For each condition, 75 axons were considered as samples. Within 10 μm of interface
Only those axons found in the analysis were included in this analysis. [Experiment of Antibody Blocking on Cell Matrix] Role of Specific FN-3 Sequence in Regulation of Axon Induction by Cell Tenascin-C
To determine the full-length tenascin-C, alternatively inherited domain fnA
Using polyclonal antibodies against D and the universal domain fn1-5
A blocking experiment was performed. Also, a monoclonal antibody that reacts within fnD of fnA-D
J1 / tn2 was also used for blocking experiments. Non-transfected and transfected BHK cells
Mixed monolayer containing 75 μl in DMEM-25 mM, KCl / 10% FCS.
Incubate for 1 hour at 37 ° C. with g / ml antibody. Apply cerebellar granule neurons to cells
The cells were clothed and cultured for 48 hours in the presence of the antibody. Then transfection and non-transfection
The neurite response at the cell interface was evaluated. Results The alternatively inherited region of tenascin-C provides a permissive axon guidance cue. fnA-D strongly promotes neurite outgrowth from various CNS neurons (
Meiners and Geller, 1997). Therefore, we also propagate fnA-D
We investigated whether it could provide guidance cues to axons. Alternatively
Or universal PLL with tenascin-C, FN-3 domain spots
Cerebellar granule neurons were cultured on coated cover glass for 48 hours. then,
The neurite response at the protein / PLL interface was analyzed. FIG. 3A shows the cerebellar condyle
The granule axons, when facing the interface between fnA-D and PLL,
Expressed strong priority. Axons (shown in red) and the fnA-D / PLL interface (f
The nA-D region is shown in green) and was visualized in black and white in FIG. 3B. Occurs on the PLL
80% or more of the neurites that have passed over to fnA-D
Less than 20% passed to the PLL. This means that about 50% of the axons generated on the PLL
Label with control fluorescein, which also crossed into BSA and vice versa
Significantly different from results obtained from neurites proliferating across the BSA / PLL interface
You. The same 50/50 cross ratio is 5 μm on the backside of the PLL coated cover glass.
I made by drawing a circle with ink, simulating the size of a protein drop
Also observed neurites proliferating across the tentative interface (data shown).
Zu). The increase in the number of neurites across fnA-D is due to the alternative
FIG. 7 illustrates providing an axon guidance cue. FIG. In contrast, the universal FN-3 domain
, Fn1-5 and fn6-8 show neurite outgrowths that differ significantly from the control.
I did not draw any response. FnA-D was identified as the expressed protein and as the largest tenascin-C splice.
Promotes neurite outgrowth as part of a subvariant (Meiners and Geller, 19
97) Therefore, we believe that in the range of natural tenascin-C,
I checked Noh. Axons with PLL and the largest or smallest Tennessee
C splice variant. Attached to purified tenascin-C
Since only a few neurons were found, only the neurite response on the PLL side of the interface was examined.
Specified. Axons consistently avoided both splice variants (FIG. 3A).
This indicates that tenascin-C (splice variant) isolated from neonatal mouse brain
Qualitatively with other results showing cerebellar granule axon deflection by spots (representing a mixture)
The same is true (Gotz et al., 1996; Dorries et al., 1996). Therefore, fn
Because the permissive inducing properties of AD are covered by other parts of the tenascin-C molecule,
Neissin C indicates that, on a molar basis, fnA-D is more inhibitory than is permissible.
Is shown. This observation reflects the dose response obtained for tenascin-C and fnA-D effects.
It also appeared in the answer curve. Inhibitory effect of both tenascin-C splice variants and fnA
Tolerable effect of -D is dose dependent, which tends to saturate at 100 and 300 nM, respectively
(Data not shown). On the other hand, the smallest tenascin-C splice ba
Liant is always more resilient than the largest Tenascin-C splice variant
So, about 10% of the axons crossed the large tenascin-C, but only 1%
-2% passed from PLL to small tenascin-C. Big tenascin C, fn
When blocked with a polyclonal antibody against AD, the percentage of neurites
The tage has dropped. This is because fnA-D contained only in large tenascin-C
Implies that the boundaries formed by the rest of the can be partially broken
are doing. fn6-8 homologous substrates (Meiners and Geller, 1997);
And small tenascin C splice variant (Chiquet and Wehrle-H
aller, 1994; Meiners and Geller, 1997) all promote neurite outgrowth
Therefore, the results of this experiment indicate that the ability to promote neurite outgrowth is not necessarily
Indicates that it may not be correlated with the ability to provide a neurite guidance cue
You. The alternatively inherited region overcomes the tenascin-C boundary and axons progress. We will then determine if the molarity of the alternatively inherited region becomes excessive,
The hypothesis that suppression of the rest of the C molecule was broken was examined. Addressing this challenge
To achieve this, we used a tank consisting of a mixture of fnA-D and small tenascin-C.
The PLL-coated cover glass with parky spots was incubated. Small tenesi
The concentration of fnA-D was 10% while the concentration of Cn was kept constant at 100 nM.
It was increased from 0 nM to 400 nM (FIG. 4A). As shown in FIG.
Only 1-2% passed to the small tenascin-C. This number is 100 nM
For small tenascin-C combined with fnA-D increased to 8-10%
Exactly the same as the percentage of neurites that crossed over to large tenascin-C (Figure 3).
there were. Axon pars over small tenascin-C and fnA-D mixtures
The percentage increases with increasing fnA-D concentration and at 300 nM fnA-D.
It was the largest. At this concentration, 60-70% of the axons result.
However, even if the concentration of fnA-D is higher than this,
Page did not increase. Thus, by itself provides an axon guidance cue
The most effective concentration of fnA-D (FIG. 3) in terms of
It is also best for overcoming a typical guiding cue. Axons are 300 nM
Than on a mixture of fnA-D and 100 nM of small tenascin-C
It passed more easily to nM fnA-D (Figure 3). This means that fnA-D
Mainly alleviates the inhibitory properties of small tenascin-C, but does not completely eliminate it
It indicates that. [0055] We also show the repressive attraction of large tenascin-C in alternatively inherited regions.
The ability to overcome the guided cue was examined. When the molar concentration of fnA-D becomes excessive,
The boundary formed by the tenascin-C splice variant is weakened (100 nM)
The maximum effect is not 300 nM, but rather a 200 nM fnA.
-D (FIG. 4B). In contrast to the case of small tenascin-C,
The low concentration of fnA-D required to break the boundaries formed by Nacin C is large.
Tenascin-C is a sequence of one fnA-D that does not contain any small tenascin-C
Represents the fact that it already contains The alternately concatenated area overcomes the CSPG boundary. Our next goal is to allow fnA-D's admissible guidance queue to
The goal was to determine whether the molecule would override the suppressive induction cue provided. I was
That effect was examined in combination with CSPG. Because CSPG is cultured
Deflecting neuronal processes within the object (Snow et al., 1990),
SPGs are often co-regulated on astrocytes (Meiners et al.,
1995; Powell et al., 1997; McKeon et al., 1991). First, PLL and mainly
Mixture of CSPG consisting of neurocan, phosphacan, versican and aggrecan
The neurite reaction at the interface formed with the compound was assessed (Table 1). As it is
Native CSPGs with lycosaminoglycan side chains are stained on SDS-PAGE gels.
It is impossible to represent points and assign accurate molecular weights (data diagram
(Not shown) in this experiment, 10 μg / m
1 CSPG mixture was used. Axons slightly avoided CSPG mixture
1-2% passed from PLL to CSPG. As in the case of Tenascin-C,
Since urones did not adhere to the CSPG mixture, axons on the CSPG side of the interface
The reaction was not assessed. Bind CSPG to fnA-D (300 nM)
Only when do the approximately 60% of the axons pass through the mixture of CSPG and fnA-D.
Was. Even higher concentrations of fnA-D increase percentage of neurites across
Did not. Table 7 fnA-D overcomes CSPG boundaries and axons advance. We have shown that the effects of fnA-D can be attributed to laminin, a potent promoter of neurite outgrowth.
-1. Laminin-1 by itself is not nearly as effective at inducing neurites.
It was not effective. In contrast to 80% of fnA-D, 45% of neurites had 3
Just crossed to 00nM laminin-1 (MLT Mercado, unpublished data)
Also, in overcoming the CSPG barrier, only about 10% of the neurites
Only passed to a mixture of CSPG and laminin-1. Laminin-1 concentration
Increasing to 1 μM increases the percentage of neurites across to 25-30%
Just added. Use a single CSPG, aggrecan, instead of the mixture
The experiment was repeated with similar results. fnA-D is an inhibitory induction cue for aggrecan
Was more effective than laminin-1 in alleviating the fnA-D
In addition, another neurite outgrowth-promoting molecule that does not provide guidance information to neurites (Meiner
s and Geller, 1997) was more effective than fn6-8 (Figure 3A). Therefore,
fnA-D specifically overcomes the boundaries formed by a variety of different CSPGs,
To move forward. Axon guidance and elongation are regulated by different sequences within fnA-D. The promotion of neurite outgrowth by fnA-D linked to an inactive substrate is indicated by fnD
(Gotz et al., 1996). So we have axons
Extension and neurite guidance are regulated by the same or different sequences in fnA-D.
Explored the question of whether it would be adjusted. For this reason, we have used axon guidance analysis
Neurite outgrowth of monoclonal antibody J1 / tn2 in both
The ability to alter the response was evaluated. This antibody promotes neurite outgrowth, especially in fnD.
Block the translocation site (Gotz et al., 1996; Gotz et al., 1997; Meiners et al.,
1999). FnA-D or fnA-D in the center of the PLL coated cover glass
Of the mixture of J1 and J1 / tn2, and the neurite reaction at the interface
Was quantified. J1 / tn2 is the neurite parsing from PLL to fnA-D
Or the percentage of axons from fnA-D to PLL
(FIG. 5A) (data not shown). Then, the PLL cover
PLL adsorbed on glass, homogeneous substrate of fnA-D, or fnA-D and J1 /
Axon outgrowth analysis was performed to quantify the outgrowth on the mixture with tn2.
. A box whisker plot of the total length of axons is shown in FIG. 5B. The box is
Surround the 25th and 75th percentiles of each distribution, divided by the median
Have been. Whiskers indicate the fifth and 95th percentiles. I expected
Thus, axons have much longer fnA-D compared to PLL, and J1 / tn
No. 2 excluded the promotion of neurite outgrowth by fnA-D. In the control experiment, fn
B monoclonal antibody tenascin 3-B (Chemicon International)
Null) did not alter the elongation or induction by fnA-D.
These results indicate that neurite induction by fnD is a sequence that promotes neurite outgrowth.
Indicates that they are regulated by different sequences. The neurite-inducing site in fnA-D was located at a specific region of the protein.
For example, fnA1-A4, which is the N end of fnA-D, and f, which is the C end of fnA-D
nB-D or a mixture of fnA1-A4 and fnB-D (300 nM each)
Cerebellar granule neurons on PLL-coated cover glass containing spots
The culture was continued for a while. Then, the neurite reaction at the expressed protein / PLL interface was evaluated.
(FIG. 6). Of axons generated on the PLL side of the PLL / fnA1-A4 interface,
Although only 20% passed to fnA1-A4, the axis formed on the fnA1-A4 side of the interface
The chords did not show any bias toward PLL or fnA1-A4 (FIG. 3).
(Comparison with neurite response at BSA / PLL control interface). Axons, on the other hand,
The priority for fnB-D was indicated. PLL to fnB-D, fnB-D
Percentage of neurites ranging from to PLL was observed for fnA-D
It was almost the same as the one (compare with FIG. 3). Therefore, fnB-D becomes fnA-D
Can be said to mimic the action of Also, an equimolar mixture of fnA1-A4 + fnB-D
Also mimicked the action of fnA-D. This indicates that the C-end of fnA-D
Provides a protrusion guidance cue, suggesting that it overcomes the inhibitory boundary formed by the N-end
I do. Next, we hypothesize that fnC provides a guiding cue for elongating axons.
I explored the theory. The rationale for this hypothesis is that cerebellar granule axons express fnC
Based on studies published to avoid deficient rodent fnA-D expressed proteins
(Gotz et al., 1996). We provide neurite guidance and neurite outgrowth
In the analytical method, fnA-D and fnA- lacking fnC (fnA-D (-) C) were used.
D-expressed proteins were compared. fnA-D or fnA-D (-) C (30
The neurite response at the 0 nM) / PLL interface was quantified (FIG. 7A). 3A
Thus, more than 80% of axons passed from PLL to fnA-D, but fnA-D
The number of axons that passed to (-) C was as small as about 25%. These data are based on fnC
Is a barrier to axonal progression formed by fnA1-A4, which is the N-terminal end of fnA-D.
This supports the hypothesis of providing an acceptable guiding cue to overcome the rear.
5). As in the case of fnA1-A4, neurites generated on fnA-D (-) C
, FnA-D (-) C or PLL. PLL cover
FnA-D or fnA-D (-) C adsorbed on the cover glass (Fig. 7B)
When neurite outgrowth analysis was performed on neurons cultured above,
The matrix was found to be equally tolerant of protrusion elongation as compared to PLL alone.
The results shown in FIG. 6 and these results indicate that neurite guidance and neurite outgrowth by fnA-D
Promotility is different for alternately inherited FN-3 domains,
implies that fnC and, for neurite promotion, are regulated via fnD
ing. FnA-D induces neurites in the area of cellular tenascin-C. Studies with purified substrates are beneficial, but many molecules are biological matrices.
Does not always predict the state of living organisms that exist in the box (Meiners and Gell
er, 1997). So we have cells that neurons would normally face.
Provides permissive axon guidance cues for fnA-D within the emerging tenascin-C
I checked my ability to do it. The largest or smallest sp. Of human tenascin-C
BHK cells transfected with rice variants (BHK-TN.L or BHK, respectively)
HK-TN. S cells) were bound to a mixed monolayer of control untransfected BHK cells.
. Incubate cerebellar granule neurons on the mixed monolayer for 48 hours, and use the transfected cells and control cells.
The reaction of neurites at the interface formed with the vesicles was assessed. FIG. 8A shows transfected cells.
A full-length antibody to tenascin-C to detect vesicles
After double immunocytochemistry with antibodies to RT97 to release
Fig. 6 is an image of a B / HHK co-culture. Axons are B from control BHK cells.
HK-TN. LHK cells passed very easily to BHK-TN. S
Avoided crossing cells. Similar results were obtained using cerebral cortical neurons
(Data not shown). This is due to axon irrespective of the splice variant that exists.
Contrast with results obtained with purified tenascin-C substrate, which always forms a barrier to chromogenesis
(Fig. 2). Next, we determined the axis at the interface formed between the transfected and control BHK cells.
The chord process response was quantified (FIG. 8B). Test cells, not matrix boundaries
BHK-TN. L or BHK-TN. S
Cells were labeled with the membrane marker PKH26. Immunocytochemical reaction and Western block
The method describes that PKH26 labeling interferes with the expression of tenascin-C by transfected cells.
(Data not shown). Axons compared to control BHK cells
And BHK-TN. Expressed preference for L cells. Axis generated on BHK cells
Approximately 70% of the cord processes are BHK-TN. LHK cells, but BHK-TN. L cell
Only 20% of the overlying axons passed to BHK cells. This is PK
Percentage of neurites to or from H26 labeled cells or from PKH26 labeled cells
BHK cells and PKH26-labeled BHK cells where the percentage was 45-50%
Neurite response observed at the control BHK / BHK interface generated between
Was quite different. Axons, on the other hand, are BHK-TN. More on S cells
Also showed preference for BHK cells. From BHK cells to BHK-TN. S cell
The percentage of neurites that crossed over (20%) was significantly lower than the control.
However, BHK-TN. Percentage of neurites from S cells to BHK cells
(60-65%) was significantly higher than the control. Therefore, BHK cells
When expressed, only small tenascin-C provides an inhibitory neurite-inducing cue.
Offer. This is because the alternatively connected region of the large tenascin-C is
The barrier formed by the abutment provides its own permissible axon guidance cue.
It suggests overcoming by offering. It is important that fnA-D provides a truly permissive induction cue within the cell matrix.
To confirm that the interference with the neurite response at the cell interface
A set of antibodies to Syn-C was tested (FIG. 9). As an antibody, full-length
Monoclonal antibodies against polyclonal antibodies against isin C, fnA-D and fn1-5
Antibody J1 / tn2 was selected. All these antibodies were found on transfected BHK cells.
Cross-reacts with a large tenascin-C splice variant. As expected
In addition, polyclonal antibody against fnA-D and monoclonal antibody J1 / tn2
Does not cross-react with small tenascin-C splice variants,
The body does not cross-react with fnA-D and the fnA-D antibody crosses fn1-5
Does not react (Meiners and Geller, 1997). The first antibody tested was a polyclonal antibody against full-length tenascin-C.
Body. In the presence of this antibody, BHK-TN. S
Or BHK-TN. The percentage of neurites that span L cells is BHK-TN
. L or BHK-TN. Percentage of neurites from S cells to BHK cells
As in the case of phage (data not shown), it is related to neurites from BHK cells to BHK cells
Was almost indistinguishable from the control values obtained (compare FIG. 8B).
This suggests that large and small tenascin-C may be produced from transfected cells.
Against the factor of BHK-TN. L and BHK-TN. S cell tolerance and suppression
This confirms that it is directly involved in the axon guidance properties. For fn1-5
The polyclonal antibody has an expressed protein corresponding to this sequence,
No view (FIG. 3), and as expected, BHK-TN. L or B
HK-TN. The percentage of neurites to S cells did not change. Then we have two antibodies, fnA-, that react in an alternatively joined region.
To investigate the effects of polyclonal and monoclonal antibodies J1 / tn2 on D
Was. Both of these antibodies are capable of converting BHK cells to BHK-TN. Axons extending to S cells
The percentage of protrusions did not change (data not shown). However
However, polyclonal antibodies against fnA-D are available from BHK-TN. Transfer to L cells
The percentage of neurites was dramatically reduced from 70% to 20%. this
In the presence of the antibody, BHK-TN. L cells are BHK-TN. Same as S cells
Axons were rejected to the same extent. Therefore, expression by transfected BHK cells
Large tenascin-C guidance queue is mapped to an alternately connected area
This indicates that the ability of fnA-D to direct neurites is
What does not appear in Syn-C (FIG. 3) but becomes apparent in BHK cell matrix
Suggests. Monoclonal J1 / tn2 was obtained from BHK cells using BHK-TN. L fine
There was no effect on the percentage of neurites extending to the bleb. Therefore,
The neurite-promoting sequence in fnA-D does not provide a guidance cue for neurites.
No. This suggests that axonals are facilitated by alternatively inherited regions of tenascin-C.
Process elongation and induction are separate events that are independently regulated on cells and inert substrates
Indicates that there is. [Discussion] The alternatively inherited FN-3 region of tenascin-C, named fnA-D,
Promotes neurite outgrowth and promotes neurite outgrowth.
fnA-D can be used as a purified expressed protein or in biological matrices.
Whether or not given to neurons as part of cell tenascin-C,
Its permissive effect is clear. Netrin (Kennedy et al., 1994; Serafi
ni et al., 1994) also have strong shadows on both axon elongation and direction.
Has a sound. However, in the case of netrin, both processes involve the same functional domain of the netrin molecule.
Coordinates through the same receptor on neurons, possibly interacting with ins
(De la Torre et al., 1997). As far as we know, fnA-
D is a different (located in the alternatively inherited FN-3 domains D and C, respectively)
A first molecule that independently promotes neurite outgrowth and induction through a sequence
Provides strong evidence that elongation and induction are separable events. The fact that human fnA-D provides a permissive induction cue is due to the fact that mouse fnA-D
Initially, there were some surprises due to published data on purified substances (Gotz et al., 1996).
It was. Mouse fnA-D has a common neurite outgrowth promoting site in fnD.
Therefore, it promotes process elongation to the same extent as human fnA-D (Gotz et al., 1996).
Are also reported to form a barrier to axons. This means that the mouse f
Several sequences unique to human fnA-D that are absent from nA-D promote neurite outgrowth
It implies that it is not related to the common axon outgrowth promoting site. This temporary
Consistent with the theory, monoclonal antibodies against neurite outgrowth promoting sites in fnD are:
It did not alter the ability of human fnA-D to induce neurites. We guide
In assays, neurites show a preference for human fnB-D, whereas murine
fnB-D was found to avoid (data not shown). Used in our analysis
Murine fnB-D expressed protein, and in a study by Gotz et al.
The mouse fnA-D expressed proteins used were both rodent c as a template.
It was obtained through PCR using DNA. However, both proteins
Appear to lack the alternatively inherited FN-3 domain C. This means that rodents
CDNA encoding the largest splice variant of tenascin-C
Does not contain this domain (LaFleur et al., 1994).
. We show that naturally occurring mutants of human fnA-D that lack fnC also
Found that they formed barriers and did not attract them. Overall consideration
These data indicate that fnC contributes to the permissive induction cue of human fnA-D.
Not only overcome the suppression-inducing cues provided by the rest of the molecule
Suggest. Conflicts with previous reports (LaFleur et al., 1994; Gotz et al., 1996)
However, the PCR data show that alternatively transcripts of mouse tenascin-C
It is possible to include fnC, albeit less likely than a mouse transcript lacking fnC.
(Dorries et al., 1996; Gotz et al., 1997). But
Does mouse fnC also provide a permissive guiding cue to axons, or alternatively
To determine if the inducibility of the region succeeded by the protein is unique to human proteins
It is very important. In contrast to the permissive induction cue provided by human fnA-D, human (see FIG. 3) and
And mice (Gotz et al., 1996; Dorries et al., 1996) Tenascin-C
Purified substrates for some splice variants are repulsive to the growing growth cone.
(Dorries et al., 1996). This growth cone repulsion property is attributed to the EGF domain.
(Gotz et al., 1996; Dorries et al., 1996). Accordingly
In the EGF domain, alternatively concatenated regions are permissive on a molar basis
Above, it is more suppressive. However, the molar concentration of fnA-D is excessively 2-3 times.
If possible, overcome the boundaries formed by various CSPGs as well as Tenascin-C
I do. Laminin-1 was not as effective, even at higher concentrations. This
This is because tenascin-C and CSPG are up-regulated on the glial scar after injury.
(McKeon et al., 1991; Pindzola et al., 1993). Te
Nasin C and CSPGs have been strongly implicated when axon regeneration fails
(Gates et al., 1996; Davies et al., 1997). Axons inhibit glial scarring
Complete recovery after CNS injury is not possible unless guided across a regulatory environment
However, this suggests a potential therapeutic role for fnA-D. We first clarified that the function of the EGF domain of tenascin-C
In contrast to the case, probably because there are molecules from the cells and their associated
Alternatively, there is a conformational restriction on cell tenascin-C (Meiners, and Geller, 1997).
Therefore, it was suggested that it was unclear in the cells. In particular, EGF domains are purified
Promotes neurite outgrowth like expressed protein (Dorries et al., 1996; Gotz
et al., 1996) show no effect on elongation within the cell tenascin-C.
(Gotz et al., 1997; Meiners and Geller, 1997). We are Tenay
Antibodies induced against the thin CFN-3 domains 6-8 and A-D were
Blocks all regulation of neurite outgrowth by Syn-C and the EGF domain does not contribute
(Meiners and Geller, 1997). Therefore, neurite promoting features
In addition to sex, the properties that form the boundaries of the EGF domain are similar in cellular tenascin-C.
Theorized that it might decay. If the EGF boundary is a biological matrix
If allowed to weaken within fnA-D, the allowed guiding cue of
It may become apparent within a splice variant. I support this hypothesis
Thus, transfected BHK cells that overexpressed small tenascin-C
Cells that overexpressed large tenascin-C
It was attractive. However, more neurites are
BHK-TN. SHK cells, but BHK-TN. L fine
Less neurites spread to the vesicles than those to purified fnA-D (Fig.
3 and FIG. 8). Similarly, in early experiments, neurites were expressed in BHK-TN. L fine
BHK cells bound with fnA-D were preferred over vesicles. This means that Tenascin
The boundary-forming properties of the EGF domain of C are partially, but not completely, within the BHK cell environment.
It indicates that it was partially excluded. Monoclones for neurite outgrowth promoting sites
The antibody is BHK-TN. L or BHK-TN. Axon pars across S cells
It did not affect the percentage. This is facilitated by fnA-D
Axonal outgrowth and elongation occur on both cell and
Indicates that the phenomenon is separable. We obtained these results using BHK cells, but showed that tenascin-C splice
Induction of neuronal processes by variants can vary depending on cell type
It is easy to imagine that there is sex. Axonal outgrowth in an alternatively spliced region
As we observed for long-promoting sites (Meiners et al., 1999), different
Cell-type specific molecules are identified for the induction of neurites within the tenascin-C molecule.
Active sites, which could mask its effects. Alternatively,
Cell-type-specific molecules provide their own neurite-inducing cues,
The induction queue may compete with or prevail over that of Tenascin-C.
No. For example, when the ratio of CSPG to fnA-D is low, neurites bend.
However, as the ratio of fnA-D increased, the neurites crossed. Therefore,
The neuronal growth regulating properties of nisin C or other matrix proteins
Can be discussed relatively. Specific tenancy by specific subset of cells
Harmonized expression of the C-splice variant regulates changes in neuronal process elongation
It may provide a moderate microenvironment to save. In summary, we report that alternatively inherited regions of human tenascin-C
Contains independent domains that promote neurite outgrowth or neurite outgrowth
Was. Axon outgrowth is promoted via alternatively inherited FN-3 domain D
The direction of extension is affected by the alternatively inherited FN-3 domain C. This
Each of these processes can be adjusted without affecting the other. this
What is important is that neurite outgrowth and neurite outgrowth are distinct fundamental mechanisms of neuronal growth.
Indicates canism. Furthermore, the induction promoting ability of fnA-D is stoichiometric.
Yes, fnA-D overcomes the inhibitory effects of both tenascin-C and CSPG
I can. Thus, fnA-D is, by itself, an axis in a suppressive environment.
It can be used as a reagent for promoting chordion growth. [Reference] 1. "Hexabrathion arms identified by tenascin-expressed proteins
Cell binding and heparin binding domains of Aukhil, I., P. Joshi, Y. Yan, and
HP Erickson. 1993. Cell-and heparin-binding domains of the hexabrachi
on arm identified by tenascin expression proteins.J. Biol. Chem. 268: 25
42-2553) 2. "Purification of hexabrathion (tenascin) from cell culture supernatant and cell contact
Separation from arrival factors ”(Aukhil, I., CC Slemp, VA Lightner, K. Nishimura,
G. Briscoe, and HP Erickson. 1990. Purification of hexabrachion (tena
scin) from cell culture conditioned medium, and separation from a cell a
dhesion factor. Matrix 10: 98-111) "Expression of Tenascin in the Developing and Adult Cerebellar Cortex" (Bartsc
h, S., U. Bartsch, U. Dorries, A. Faissner, A. Weller, P. Ekblom, and M.
Schachner. 1992. Expression of tenascin in the developing and adult cer
ebellar cortex. J. Neurosci. 12: 736-749) "Tenacinin C in peripheral nerve morphogenesis" (Chiquet, M. and B. Wehrle-
Haller. 1994. Tenascin-C in peripheral nerve morphogenesis. Perspec. Dev
Neurobiol. 2: 67-74) 5. "A cell surface receptor, tenascin-C, interacts with annexin II.
Induced mitogenesis, cell migration and loss of localized adhesions "(Chung, C.-Y., J.
E. Murphy-Ullrich, and HP Erickson. 1996. Mitogenesis, cell migration,
and loss of focal adhesions induced by tenascin-C interacting with its
Cell surface receptor, annexin II. Mol. Biol. Cell 7: 883 -892) "Cytotactin and its proteoglycan ligand are found in the early postnatal body of mice.
Mark structural and functional boundaries within the sexual sensory cortex. (Crossin,
KL, S. Hoffman, SS Tan, and GM Edelman. 1989. Cytotactin and its
proteoglycan ligand mark structural and functional boundaries in somatos
ensory cortex of the early postnatal mouse. Dev. Biol. 136: 381-392) "Regeneration of adult axons in white matter tracts of the central nervous system" (Davies, SJ, MTF
itch, SP Memberg, AK Hall, G. Raisman, and J. Silver. 1997. Regenera
tion of adult axons in white matter tracts of the central nervous system
Nature 390: 680-683) "Netrin-1 Gradient Regulated by Netrin Receptor DCC
Direction of the retinal growth cone in the umbrella ”(de la Torre, JR, VH Hopker, GL Ming,
MM Poo, M. Tessier-Lavigne, Hemmati-Brivanlou, and CE Holt. 1997. T
urning of retinal growth cones in a netrin-1 gradient mediated by the ne
trin receptor DCC. Nueron 19: 1211-1244) "Recombinant tags for neuronal cell adhesion, growth cone induction and neuronal polarity.
Outstanding Effects of the Nasin Domain "(Dorries, U., J. Taylor, Z. Xioa, A.
Lochter, D. Montag, and M. Schachner. 1996.Distinct effects of recombin.
ant tenascin domains on neuronal cell adhesion, growth cone guidance, an
d neuronal polarity. J. Neurosci. Res. 43: 420-438) "Prominent extracellular matrix proteins,
Nasin "(Erickson, HP and MA Bourdon. 1989. Tenascin: an extracel
lular matrix protein prominent in specialized embryonic tissues and tumo
rs. Annu. Rev. Cell Biol. 5: 71-92) "Tenacin, laminin and fibronectin produced by cultured cells" (Er
ickson, HP and Briscoe, G. (1995) Tenascin, laminin, and fibronectin
produced by cultured cells.In: Extracellular matrix: a practical approa
ch. MA Haralson and JR Hassell, eds. Oxford Univ. press, Oxford.pp.
187-198) 12. "J1 / tenascin is a repulsive substrate for central nervous system neurons.
You. (Faissner, A. and J. Kruse. 1990. J1 / tenascin is a repulsive subs
trate for central nervous system neurons. Neuron 5: 627-637) 13. "Tenacin C domain in cell adhesion, anti-adhesion and promotion of neurite outgrowth"
(Fischer, D., M. Brown-Ludi, T. Schulthess, and R. Chiquet
-Ehrismann. 1997. Concerted action of tenascin-C domains in cell adhesio
n, anti-adhesion and promotion of neurite outgrowth.J. Cell Sci. 110: 15
13-1522) "For localization and tracking of endothelial cells in the examiner and in vivo.
Features and Effectiveness as Labels ”(Ford, JW, TH3. Welling, JC Stanley
, and LM Messina. 1996, PKH26 and 125I-PKH95: characterization and eff
icacy as labels for in vitro and in vivo endothelial cell localization a
nd tracking. J. Surg. Res. 62: 23-28) "In the examiner, chondroitin sulfate in the neostriatum of injured adult mice
Roteoglycan and tenascin: dopamine neuron attachment and
Projection extension ”(Gates, MA, H. Fillmore, and DA Steindler. 1996. Chondroit
in sulfate proteoglycan and tenascin in the wounded adult mouse neostria
tum in vitro: dopamine neuron attachment and process outgrowth.J. Neuro
sci. 16: 8005-8018) "Filapodia at the laminin / fibronectin interface within the examiner
Initiate the sensory neuron growth cone option. (Gomez, TM and PC L
etourneau. 1994. Filopodia initiate choices made by sensory neuron growt
h cones at laminin / fibronectin borders in vitro. J. Neurosci. 14: 5959-59
72) 17. "Tenacinin C is a distinct adhesive site for neurons, an anti-adhesive site
And a site for promoting neurite outgrowth. (Gotz, B., A. Scholze, A. Clement
, A. Joester, K. Schutte, F. Wiggcr, R. Frank, E. Spiess, P. Ekblom, and
A. Faissner. 1996. Tenascin-C contains distinct adhesive, anti-adhesive
, and neurite outgrowth promoting sites for neurons.J. Cell Biol. 132: 6
81-699) 18. "Tenacin C synthesis and its effect on axonal growth in the developing murine cortex" (Gotz
, M., J. Bolz, A. Joester, and A. Faissner. 1997. Tenascin-C synthesis a
nd influence on axon growth during rat cortical development.Eur.J. Neu
rosci. 9: 496-506) 19. "Tenacin Mr220,000 isoform expression correlates with corneal cell migration.
is there. (Kaplony, A., DR Zimmermann, RW Fischer, BA Imhof, BF Od
ermatt, KH Winterhalter, and L. Vaughan. 1991. Tenascin Mr 220 000 iso
form expression correlates with corneal cell migration.Developmemt 112:
605-614) 20. "Netrin is a diffusive chemotactic factor for commissural axons in the embryonic spinal cord
. (Kennedy, TE, T. Serafini, JR de la Torre, and M. Tessier-Lavig
ne. 1994. Netrins are diffusible chemotropic factors for commissural axo
ns in the embryonic spinal cord. Cell 78: 425-435) "Platelet-derived growth factor BB is an alternative to murine tenascin
Cloning and characterization of isolated isoforms "" Tenascin mRNA in arterial smooth muscle cells
Significantly enhance the expression of the inherited mutant. (LaFleur, DW, JA Fagin, J
.S. Forrester, SA Rubin, and BG Sharifi. 1994. Cloning and character
Platelet-deri: ization of alternatively spliced isoforms of rat tenascin.
ved growth factor-BB markedly stimulates expression of spliced variants
of tenascin mRNA in arterial smooth muscle cells.J. Biol. Chem. 269: 207
57-20763) 22. "Cell attachment to frozen sections of injured brains of adult mice"
Effect and lectin perturbation of extracellular matrix molecules associated with injury "(Laywell, E
.D., P. Friedman, K. Harrington, JT Robertson, and DA Steindler. 199
6. Cell attachment to frozen sections of injured adult mouse brain -Effe
cts of tenascin antibody and lectin perturbation of wound-related extrac
ellular matrix molecules. J. Neurosci. Meth. 66: 99-108) "Regulation of growth cone motility by lower layer binding molecules and cytoplasm [Ca2 +]" (Le
tourneau, PC, DM Snow, and TM Gomez. 1994.Regulation of growth co
ne motility by substratum bound molecules and cytoplasmic [Ca2 +]. Prog.
Brain Res. 103: 85-98) 24. "Autoradiographic analysis of acidic amino acids in distinguishing cerebellar granule cell cultures.
Positioning by Fi and depolarization release "(Levi, G., F. Aloisi, MT Ciotti
, and V. Gallo. 1984. Autoradiographic localization and depolarization-i
nduced release of acidic aminoacids in differentiating cerebellar granul
e cell cultures. Brain Res. 290: 77-86) 25. "J1 / tenascin in a substrate-bound and soluble state is responsible for neurite outgrowth.
Represents the opposite effect. (Lochter, A., L. Vaughan, A. Kaplony, A. Prochian
tz, M. Schachner, and A. Faissner 1991.J1 / tenascin in substrate-bound a
nd soluble form displays contrary effects on neurite outgrowth.J. Cell
Biol. 113: 1159-1171) 26. "Reduced neurite outgrowth in a model of glial scar after CNS injury is
There is a correlation between the expression of inhibitory molecules on refractory astrocytes. (McKeon, RJ, R.
C. Schreiber, JS Rudge, and J. Silver. 1991.Reduction of neurite outg
rowth in a model of glial scarring following CNS iniury is correlated wi
th the expression of inhibitory molecules on reactive astrocytes.J. Neu
rosci. 11: 3398-3411) 27. "Long and short splice variants of human tenascin are
Differentially control protrusion extension. (Meiners, S. and HM Geller. 1997. Long
and short splice variants of human tenascin differentially regulate neur
ite outgrowth. Mol. Cell. Neurosci. 10: 100-116) "A separate subset of tenascin / CSPG-rich astrocytes was
Limit neuronal growth within. (Meiners, S., EM Powell, and HM
Geller. 1995.A distinct subset of tenascin / CSPG-rich astrocytes restri
cts neuronal growth in vitro. J. Neurosci. 15: 8096-8108) 29. "Promoting neurite outgrowth by alternatively inherited regions of tenascin-C
Affected by type-specific bindings. (Meiners, S., EM Powell, and HM
Geller. 1999. Neurite outgrowth promotion by the alternatively spliced
region of tenascin-C is influenced by cell type-specific binding.
Biology 18: 75-87) 30. "5 (6) carboxyfur of embryonic neurons cultured on astrocyte monolayers
Selective Labeling with Olesein Diacetate (CFDA) "(Petroski, RE
and HM Geller. 1994.Selective labeling of embryonic neurons cultured
on astrocyte monolayers with 5 (6) -carboxyfluorescein diacetate (CFDA).
Neurosci. Meth. 52: 23-32) 31. "In the dorsal root entry zone of the spinal cord during development and after nerve root and sciatic nerve lesions
Putatively suppressed extracellular matrix molecules ”(Pindzola, RR, C. Doller, a
nd J. Silver. 1993.Putative inhibitory extracellular matrix molecules a
t the dorsal root entry zone of the spinal cord during development and a
fter root and sciatic nerve lesions. Dev. Biol. 156: 34-48) 32. "Proteoglycans provide axonal guidance at the astrocyte border
. (Powell, EM, JW Fawcett, and HM Geller. 1997. Proteoglycans p
rovide neurite guidance at an astrocyte boundary.Mol.Cell Neurosci. 27
-42) 33. "Analysis of astrocyte regulatory cues in neuron induction and process extension" (Po
well, EM and HM Geller. 1999.Dissection of astrocyte-mediated cues
in neuronal guidance and process extension. Glia 26: 73-83) 34. "Netrin is a homologous axonal growth-promoting protein on C. elegans UNC-6.
Define the family of quality. (Serafini, T., TE Kennedy, MJ Galko, C. Mirzaya
n, TM Jessell, and M. Tessier-Lavigne. 1994. The netrins define a fami
ly of axon outgrowth-promoting proteins homologous to C. elegans UNC-6.
Cell 78: 409-424) 35. "The role of forebrain astrocytes in development, impaired regeneration, and induced regeneration after transplantation
Change (Smith, GM, RH Miller, and J. Silver. 1986. Changing role of
forebrain astrocytes during development, regenerative failure, and induc
ed regeneration upon transplantation. J. Comp. Neurol. 251: 23-43) "Sulfated proteoglycans in the astrocyte barrier are responsible for axonal outgrowth in the examiner.
Inhibits elongation. (Snow, DM, V. Lemmon, DA Carrino, AI Caplan,
and J. Silver. 1990.Sulfated proteoglycans in astroglial barriers inhib
It neurite outgrowth in vitro. Exp. Neurol. 109: 111-130) 37. "Chondroitin sulfate proteoglycans are used by retinal ganglion cells to grow.
Direction may be affected. (Snow, DM, M. Watanabe, PC
Letourneau, and J. Silver.1991.A chondroitin sulfate proteoglycan may
influence the direction of retinal ganglion cell outgrowth. Development
113: 1473-1485) 38. "Boundaries defined by adhesion molecules during cerebral cortex development: J1 / mouse
Tenascin glycoprotein in the barrel field of the somatosensory cortex ”(Steindler, DA, NG
Cooper, A. Faissner, and M. Schachner. 1989. Boundaries defined by adh.
esion molecules during development of the cerebral cortex: the J1 / tenasc
in glycoprotein in the mouse somatosensory cortical barrel field. Dev. B
iol. 131: 243-260) 39. "In testers, we look at the growing axons and the different substrate affinities of migrating cells.
Analytical Methods to Test "(Vielmetter, J., B. Stolze, F. Bonhoeffer, and C.
A. Stuermer. 1990. In vitro assay to test differential substrate affinit
ies of growing axons and migratory cells. Exp. Brain Res. 81: 283-287) "Tenacinin C expression and axons following injury to the dorsal column of adult rats.
Germination "(Zhang, Y., JK Winterbottom, M. Schachner, AR Lieberman, and
PN Anderson. 1997. Tenascin-C expression and axonal sprouting followin
g injury to the spinal dorsal columns in the adult rat.J. Neurosci. Res
49: 433-450) Example 2 We have an alternating fibronectin type called fnA-D.
A region of tenascin-C containing only the -3 domains AD is used by itself.
Dramatically increases neurite outgrowth from various CNS neurons in culture
I discovered that. In addition, the fnA-D protein is not available after injury to the central nervous system.
Of chondroitin sulfate proteoglycan which may suppress regrowth
It can overcome the suppression effect. We believe that the fnA-D region of tenascin-C
Found that, when used by itself, promotes: (1) Axon growth (2) Axon extension across inhibitory boundaries To further illustrate these phenomena, we used overlapping synthetic peptides.
The neurite outgrowth-promoting sites in fnD were identified by two 15 amino acids called D4 and D5.
No acid sequence was determined. Antibodies to D5 were obtained from cerebellar granule neurons, fnD
And promotion of neurite outgrowth by tenascin-C. This means that this pepti
Indicates that the sequence is functional in the range of the natural molecule. Next we have 8 amino
Acid, VFFDNFLK (SEQ ID NO: 1) to limit the neurite outgrowth promotion site,
By testing shorter synthetic peptides, the overlapping region of D4 and D5
evaluated. This amino acid is AA1646-1 of human tenascin C-cDNA.
653 (“Human tenascin gene, structure of 5 ′ region, knowledge of transcription control sequence”
Gherzi, R. Carnemolla, B., Siri, A., Ponassi, M., Baiza, E.
and Zardi, L. Human tenascin gene.Structure of the 5'-region, identific
ation, and characterization of the transcription regulatory sequences.J
Biol. Chem. 270, 3429-3434. 1995; MEDLINE 95155442; Genbank ACCESSION:
X78565). Of these, "FD" and "FV" are Genbank
And is conserved within the tenascin-C sequence from all species available. "F
The hypothesis that "D" and "FV" are critical for interaction with neurons was investigated.
For comparison, we have identified recombinant fnD protein and "FD" and "FV"
Each was tested with synthetic peptides changed to "SA" and "SS". These molecules are sudden
Did not promote elongation. This indicates that the conserved amino acids are located at the active site in fnD.
Suggests that it is necessary for formation. Our conclusion is that the growth-promoting region of fnA-D is within fnD,
The identified minimal region is the human tenascin C-, which is included in GenBank.
Seven amino acids consisting of AA1647-153 of cDNA. Example 3 We also note that VFDNFVLK is a major component found in the central nervous system following injury.
Axonal outgrowth of chondroitin sulfate proteoglycan (CSPG), a novel inhibitory molecule
It was investigated whether it can be used as a reagent that overcomes the elongation-inhibition property. New
-Ron does not proliferate when contacted with CSPG. When mixed with CSPG,
Peptides significantly enhance elongation on proteoglycans, and neurite outgrowth
It was more effective than laminin-1 or L1-Fc, which are long promoting molecules. But
Thus, fnA-D and its smaller derivatives are capable of regenerating neurons following CNS injury.
It may be useful as part of a regeneration reaction mixture that promotes length. Example 4 We have a receptor for D5 that regulates neurite outgrowth promoting action
I hypothesized that. For tenascin-C, extracellular matrix
Β1 integrin, which regulates the signal to the case, may act as a receptor
I already know. For this reason, we proposed tenaxin elongation promoting tenascin-C peptide.
The potential role of integrins as receptors for de VFDNFVLK
Examined. We found that a commercially available blocking antibody to the β1 integrin chain was
Of neurite outgrowth by VFDNFVLK and fnA-D
It has been found to eliminate the use. This indicates that β1 integrin binds to this sequence.
Support the role of neuron receptors. Next, we searched for an α-chain partner for the β1 integrin chain. α7 in
Tegulin knockout rats have a defect in the tenascin-C-rich intertenus junction
To show, we show that the α7 integrin chain is a neurite outgrowth by VFDNFVLK.
The hypothesis of being a partner for β1 in promoting elongation was tested. α7 integri
Antibodies to neurite outgrowth by VFDNFVLK and fnA-D
Was eliminated. These results indicate that monoclonal α recognizing the VFDNFVLK sequence
7 cannot explain. Because this antibody is
Did not cross-react with the peptide. Therefore, α7β1 integration
Phosphorus regulates the promotion of neurite outgrowth by VFNFVLK and fnA-D.
It is a uron receptor. Our data show that this receptor is laminin-1
Is the first evidence for modulating the response to non-matrix molecules
And the first role of the α7β1 integrin receptor in neurons
Functional data. Peripheral neurons that regenerate processes after trauma are associated with α7β1 in
By dramatically increasing the expression of tegulin, environmental factors such as tenascin-C
It is possible to react with an acceptable substrate molecule. Example 5 In addition to inhibiting growth, the injured area of the central nervous system is not
Form a boundary. This property is caused by upregulation in proteoglycan synthesis.
It has been attributed. We have created an analytical method for boundary formation, and
That neurons can overcome proteoglycan boundaries
discovered. Tenascin-fnA-D protein without fnC has this property.
Therefore, the fnC region is essential for this function. Therefore, crossing the permissible boundary
Capabilities are likely to be in fnC. Example 6 We Promote the Regrowth of Injured Axons in the Human Central Nervous System
Therefore, it is proposed to use fnA-D or a small region thereof for treatment. This
Protein can be delivered to the area of the spinal cord lesion for therapeutic purposes. For example,
The smallest, potentially potent peptide is used as an infusate via a catheter.
It can be supplied directly into the metamorphic site. Nutrients to survive neurons or
In combination with other drugs, such as caspase inhibitors, and other drugs that promote neuronal growth
To supply the peptide. This is because proteoglyca in the nervous system
It may be performed in combination with a treatment that limits the expression of the protein. Alternatively, the cells in the lesion area may be genetically modified as follows to express fnA-D:
A child feeding technique can be used. Astrocytes or other glia within the lesion
The cells are made to express a transgene containing the fnA-D region or a small region thereof. fn
A-D other transgenes promote glial fibrillary acid gene on astrocytes
Or other suitable enhancer for the expression of other cell types in the area of injury
Under the control of cell type specific promoters. These components are
Inject into the lesion area. As a viral vector, glia
Adeno-associated virus or lenti that expresses genes in vesicles or fibroblasts
A virus may be used. This construct is used to limit expression to astrocytes,
nA-D may be placed under the control of a GFAP promoter. References of Interest “Expression of oligodendrocyte specific gene in mouse brain: within adeno-associated virus
Use of myelin-forming cell type-specific promoters "(Chen H, McCarty DM, Bruce AT
, Suzuki. Oligodendrocyte-specific gene expression in mouse brain: use o
fa myelin-forming cell type-specific promoter in an adeno-associated vi
rus. J. Neurosci. Res. 1999 Feb 15; 55 (4): 504-13) "Stable genes for the nervous system using non-primate lentiviral vectors.
Metastasis ”(Mitrophanous K, Yoon S, Rohll J, Patil D, Wilkes F, Kim V, Kings
man S, Kingsman A, Mazarakis N. Stable gene transfer to the nervous syst
em using a non-primate lentiviral vector.Gene Ther. 1999 Nov; 6 (11): 180
8-18) Example 7 We also expressed α7β1 integrin receptor in neurons.
Gene delivery to enhance neuron response to fnA-D.
And suggest. This can be a replication-inactive Sindbis virus vector, or
It can be achieved with a herpes virus vector that is neurotropic. References of Interest “The 51 kilobase HSV-1 plasmid vector contains the helper virus
Can be packaged using a system that does not support the expression in the mouse brain.
To (Wang X, Zhang GR, Yang T, Zhang W, Geller AI Fifty-one kil
obase HSV-1 plasmid vector can be packaged using ahelper virus-free syst
em and supports expression in the rat brain.Biotechniques 2000 Jan; 28
(1): 102-7) "In a single major cortical neuron using a replication-ineffective HSV vector
Expression of multiple proteins "(Coopersmith R, Neve RL Expression of multiple pr
oteins wthin single primary cortical neurons using a replication deficie
nt HSV vector. Biotechniques 1999 Dec; 27 (6): 1156-60) “Intraneurons using recombinant adenovirus with neuron specific promoter
Efficient gene transfer and long-term expression in plants "(Navarro V, Millecamps S, Geoffro
y MC, Robert JJ, Valin A, Mallet J, Gal La Salle GL Efficient gene trans
fer and long-term expression in neurons using a recombinant adenovirus w
Gene Ther. 1999 Nov; 6 (11): 1884-92) [Brief Description of the Drawings] FIG. 1 shows a multi-domain structure of human tenascin-C. This figure shows
Adapted from Aukhil et al. (1993, J. Biol. Chem. 268: 2542-2553)
It was made. The N-termini of the three arms are joined to form a trimer, and
The two trimers are linked by a disulfide bond to form a hexamer.
Each arm relies on 14 EGF domains, alternative RNA splicing
8 to 15 FN-3 domains and a single fibrinogen domain
You. The universal FN-3 domains (fn1-5 and fn6-8) are
Present in the Isin C splice variant. In addition, the biggest tenascin C
The price variant comprises seven alternatively inherited FN-3 domains (A1, A
2, A4, B, C and D, i.e. fnA-D), but the shortest
These are not present in the new splice variants. FIG. 2 is a schematic diagram showing an axon guidance assay. PLL coated hippo
-Drop a drop of the protein solution of interest onto the center of the glass
After that, the excess protein solution was washed away to create the protein / PLL interface
. On this cover glass, cerebellar granule neurons are cultured for 48 hours, and then
Using an antibody against the protein in the drop and an antibody against the neurofibrillary tangle
Double immunocytochemical reactions were performed. And for the axons that occur on the PLL,
In addition, the neurites at the interface are compared
The reaction was analyzed. The following criteria were established for this induction potency assay. interface
Consider only a single non-bundle axon within 10 μm of
Count only the axons, not the axons where the cell body lies on the interface.
Did not. [FIG. 3] The alternatively inherited region of tenascin-C is the permissible axon guidance cue.
To provide (A) Cerebellar granule neurons were transformed with fn1-5, fnA-
D, fn6-8, large tenascin-C (TN.L) or small tenascin-C
Incubated for 48 hours on PLL-coated cover glass containing (TN.S) spots
. Axons from the PLL to the protein spot, and vice versa
The percentage of neurites that crossed over was assessed. Bars represent mean ± SEM
(N = 4). In a control experiment, 51 ± 4% of the axons resulted from the PLL
Resen labeled BSA control and 50 ± 2% from BSA to PLL
Crossed over. The neurite reaction at the fn1-5 or fn6-8 / PLL interface is
, Did not differ significantly from the control. Axons from PLL to fnA-D
The percentage of protrusions (asterisk in the figure) is significantly higher than the control,
And the percentage of neurites from fnA-D to PLL (double asteris
Was significantly lower than the control (p <0.05, Student-Newman-Keuls
test). In contrast to fnA-D, large or small tenas
The percentage of neurites crossing over to C (cross) was significantly lower than the control.
(P <0.05, Student-Newman-Keuls test). Polyclones for fnA-D
Antibody (pAb) is the percentage of neurites across large tenascin-C
Was further reduced (double cross). This reduction is significant (p <0.05, Stude
nt-Newman-Keuls test). (B) For full length tenascin-C, fnA-
To detect D spots, a polyclonal antibody was added, followed by fluorescein.
A double immunocytochemical reaction was performed using the second antibody. In addition, neurons are detected.
In order to generate a second antibody conjugated with rhodamine after the monoclonal antibody RT97
Using . Axons on either side of the PLL / fnA-D interface are
Priority. Bars are 10 μm. FIG. 4 fnA-D overcomes the border of tenascin-C and axons progress
It indicates that. Small tenascins C and fnA-D (A) or large tenascins
PLL coated cover comprising protein spot consisting of Syn C and fnA-D (B)
-Cerebellar granule neurons were cultured on glass for 48 hours (n = 3). Tenay
While keeping the concentration of syn C constant at 100 nM, the concentration of fnA-D was increased to 100 nM.
M (ratio of fnA-D to tenascin-C is 1: 1) to 400 nM (ratio, 4: 1)
). Axons from PLL to tenascin C / fnA-D spot
Increased with increasing concentration of fnA-D. Small tenesi
300 nM of fnA-D for the protein C and for large tenascin-C
200 nM fnA-D was observed to be the most effective. FIG. 5. The neurite outgrowth promoting site in fnD does not regulate neurite outgrowth.
To indicate that (A) fnA-D, fnA-D and monoclonal antibody (mAb) J
1 / tn2 or fnA-D and monoclonal antibody tenascin 3-
B. On a PLL coated cover glass containing spots of the mixture of mAb3-B,
Cerebellar granule neurons were cultured for 48 hours (n = 4). J1 reacts in fnD
/ Tn2 changes the percentage of neurites from PLL to fnA-D
And changes by mAb3-B reacting in fnB
I never did. (B) On PLL coated cover glass or fnA-D
Or a mixture of fnA-D and J1 / tn2 or mAb3-B was adsorbed
Forty-eight hours, granule neurons in the cerebellum grow out of axons on PLL-coated cover glass
Can be extended. Box-whisker plot of axon total length distribution
Then, one of four representative experiments is shown. The boxes are the 25th of each distribution and
Surrounds the 75th percentile and is bisected by an intermediate number. Whisker is 5th
The 95th and 95th percentiles are shown. fnA-D has more axons than PLL alone
Although J1 / tn2 promoted neurite outgrowth, J1 / tn2 eliminated the neurite outgrowth promoting properties of fnA-D.
Removed. mAb3-B had no effect. FIG. 6 shows that axon guidance is locally restricted to the C-terminal of fnA-D.
And fnA1-A4, fnB-D, or fnA1-A4 and fnB-D
Cerebellar granules on a PLL-coated cover glass containing spots of the mixture with
Were cultured for 48 hours (n = 3). fnA1-A4 is the axis generated on the PLL
An inhibitory boundary was formed for the chords. of axons generated on fnA1-A4
The response was somewhat random (dashed lines indicate neurites at the BSA / PLL interface
Indicates that the reaction is random). On the other hand, it is produced on PLL or fnB-D.
Axons that have been shown showed a preference for fnB-D. Also, fnA1-A4 and
Since the mixture with fnB-D also imitated the action of fnA-D, fnB-D
The boundary formed by A1-A4 was overcome. FIG. 7 shows that fnC is involved in regulating neurite guidance. (A)
PLL-coated cover gas containing fnA-D or fnA-D (-) C spots
Cerebellar granule neurons were cultured on the lath for 48 hours (n = 3). Interface PLL
Lateral axons preferred fnA-D, but avoided fnA-D (-) C. Interface
The neurites on the protein side favored fnA-D, but with respect to fnA-D (-) C.
Showed a random response (dashed line). (B) fnA-D or fnA-D (
-) 4 cerebellar granule neurons were placed on C-adsorbed PLL-coated cover glass.
The cells were cultured for 8 hours. One of four representative experiments is shown. fnA-D (FnA-D)
And fnA-D (-) C both promote neurite outgrowth compared to PLL
However, the distribution of neurite lengths on fnA-D and fnA-D (-) C was the same.
Was. FIG. 8: fnA-D induces neurites within cellular tenascin-C
To do so. (A) BHK cells and BHK-TN. L or BHK-
TN. Cerebellar granule neurons were cultured for 48 hours on a mixed monolayer of S cells. Complete
For full length tenascin-C, a polyclonal antibody followed by fluorescein
Double immunocytochemical reactions were performed with the in-conjugated second antibody. Also monochrome
The antibody RT97 was used followed by a second antibody conjugated with rhodamine. Axon
Shows that BHK-TN. LHK cells, but BHK-TN. S cells
avoided. Bars are 12 μm. (B) Quantification of the neurite response at the cell interface
(N = 4). In control experiments, 45-50% of the neurites were BHK cells
From BHK cells labeled with PKH26 and vice versa. BHK fine
BHK-TN. The percentage of neurites across L cells is higher than the control.
BHK-TN. From L cells to BHK cells
Were significantly lower (double asterisk) (p <0.05, Stude
nt-Newman-Keuls test). In contrast, BHK-TN. Axons over S cells
Is much lower than the control (cross), and the percentage
The content was quite high (double crosses) (p <0.05, Student-Newman-Keuls
test). FIG. 9 shows neurite outgrowth in the presence of the tenascin-C antibody. (A) Complete
In the presence of full length polyclonal antibodies to tenascin-C, BHK cells
And BHK-TN. L or BHK-TN. On a mixed monolayer with S cells
Granular neurons were cultured for 48 hours. The antibody is derived from BHK cells and BHK-TN
. Reduce the percentage of neurites to L cells from about 70% to 50% (
Asterisk), BHK-TN. Of neurites to S (BHK-TN.S) cells
Increased from about 20% to 50% (cross) (p <0.05, Student
-Newman-Keuls test). (B) In addition, the port for fn1-5 or fnA-D
With a clonal or monoclonal antibody (mAb) J1 / tn2
In BHK cells and BHK-TN. Culture neurons on a monolayer mixed with L cells
Was. The fn1-5 antibody is BHK-TN. Axon percentages across L cells
And J1 / tn2 had no effect. fnA-D antibody
Significantly reduced the percentage of axons that crossed to about 20% (double
(Asterisk) (p <0.05, Student-Newman-Keuls test). [Procedure amendment 2] [Document name to be amended] Drawing [Item name to be amended] All drawings [Correction method] Change [Contents of amendment] FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C R, CU, CZ, DE, DK, DM, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, K Z, LC, LK, LR, LS, LT, LU, LV, MA , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG , US, UZ, VN, YU, ZA, ZW (71) Applicant 335 George Street, P.M.             O. Box 2688, New Brunsw             ic, NJ 08903, U.S.A. S. A. (72) Inventor Herbert Geller M.             United States, New Jersey             08094, Highland Park, South Africa             Delide Avenue 44 (72) Inventor Sally Mayners             United States, New Jersey             08854, Piscataway, Bed Four             De Cote 117 F term (reference) 4H045 AA10 BA15 CA40 EA21 EA50

Claims (1)

Claims 1. An eight amino acid sequence V defined by a one-letter amino acid code
A peptide consisting of FDNFVLK, the peptide consisting of not more than 75 amino acids.