WO2000066628A1 - Neurite outgrowth and guidance by tenascin-c - Google Patents

Abstract

We developed an assay to quantify neurite behavior at sharp substrate boundaries and found that neurites demonstrated a strong preference for fnA-D when given a choice at a poly-L-lysine/fnA-D interface. Furthermore, neurites preferred cells that over expressed the largest but not the smallest tenascin-C splice variant when given a choice between control cells and cells transfected with tenascin-C. The permissive guidance cues of large tenascin-C expressed by cells were mapped to fnA-D. We demonstrate that neurite outgrowth and guidance are facilitated by distinct sequences within fnA-D. Hence, neurite outgrowth and neurite guidance mediated by the alternatively spliced region of tenascin-C are separable events which can be independently regulated.

Description

Neurite Outgrowth and Guidance by Tenascin-C

This work was supported by NIH R01 NS24168 to H.G and NIEHS Exploratory Research Award RQ1610 to S.M.

This application incorporates material from and cites priority of U.S. provisional

application serial no. 60/132,137 filed May 1, 2000.

BACKGROUND OF THE INVENTION

Tenascin-C has been implicated in regulation of both neurite outgrowth and neurite guidance. We have previously shown that a particular region of tenascin-C has powerful neurite

outgrowth promoting actions in vitro. This region consists of the alternatively spliced fibronectin

type-Ill (FN-III) repeats A-D and is abbreviated fnA-D.

Development of the nervous system is absolutely dependent upon targeted growth of

axons and dendrites. Not only must neuronal processes elongate to reach their correct destination (neurite outgrowth), but they must navigate in the proper direction (neurite guidance). Both

neurite outgrowth (Smith et al., 1986) and neurite guidance (Letourneau et al., 1994) are thought

to be regulated by astrocyte-derived surface molecules. Amongst these molecules is tenascin-C, an extracellular matrix protein which is transiently expressed at the boundaries of migratory pathways in the developing cortex (Steindler et al.. 1989) and is re-expressed on glial scars in the adult central nervous system (CNS) (McKeon et al, 1991 ; Laywell et al., 1996; Lochter et al, 1991). Based upon its localization, tenascin-C was originally thought to form barriers to advancing neuronal processes by stunting their outgrowth and/or deflecting them elsewhere

(Steindler et al., 1989). However, functional studies in vivo (Gates et al, 1996; Gotz et al., 1997; Zhang et al., 1997) and in vitro (Faissner and Kruse, 1990; Lochter et al., 1991 ; Meiners and

Geller, 1997) have demonstrated that tenascin-C can provide permissive as well as inhibitory cues for neuronal growth.

Tenascin-C is not a single molecule, but is instead a family of alternatively spliced

variants with potentially diverse actions (Chung et al., 1996; Gotz et al., 1997; Meiners and Geller, 1997). Tenascin-C splice variants differ only in their number of FN-III domains; for example, the largest splice variant of human tenascin-C has seven alternatively spliced FN-III domains (designated fnA-D. Figure 1) that are missing in the smallest splice variant. Phases of increased cell migration and axonal growth in the developing CNS have been closely correlated

with expression of large but not small tenascin-C (Crossin et al., 1989; Steindler et al., 1989;

Kaplony et al.. 1991 ; Bartsch et al., 1992), suggesting that fnA-D might facilitate cell and neurite motility during embryogenesis. Our own structure-function studies of tenascin-C splice variants demonstrated that fnA-D avidly promoted neurite outgrowth in vitro from a variety of neuronal

types, both by itself as a bacterial expression protein and as part of large tenascin-C (Meiners and

Geller, 1997; Meiners et al.. 1999).

SUMMARY OF THE INVENTION

It is an object of the invention to develop a method of stimulating axonal and/or dendritic

growth and guidance. Other objects and advantages of the invention will become apparent to

those skilled in the art from the accompanying description of the invention. In one general aspect, the invention is a peptide comprising the 8-amino acid sequence

VFDNFVLK. as defined by the one-letter amino acid code, said peptide consisting of not more

than 75 amino acids in particular embodiments adapted for situations where it is more

appropriate to use smaller peptides, the peptide consists of not more than 50 amino acids, more

preferably not more than 20 amino acids, even more preferably not more than 10 amino acids.

The 8-amino acid peptide unlinked to other amino acids is itself an invention.

In a related aspect, the invention is a peptide comprising a tenascin-C region selected

from the group consisting of fnA-D, fnD. and fnC, said peptide free of any tenascin-C region,

other than the selected tenascin-C region, exceeding 100 amino acids in length. (More preferably

said peptide is free of any tenascin-C region, other than the selected tenascin-C region,

exceeding 10 amino acids in length).

In another aspect, the invention is a method of stimulating axonal or dendritic growth

and/or guidance, said process comprising administering a peptide described in the preceding 2

paragraphs to a neuron. In one embodiment, the axon or dendrite is in a human nervous system.

For example, the peptide is delivered to the spinal cord. In one delivery mode the peptide is

delivered by infusion. In a related aspect of the invention, a vector is adminstered to an area of

injury to the nervous system, the vector being nucleic acid comprising a base sequence coding

for the peptide. In an alternative approach, nucleic acid molecule is in a virus at the time of

administration.

In a related aspect, the invention is a method of stimulating axonal or dendritic growth

or guidance, said method comprising administering a peptide to an axon or dendrite. said peptide

being at least 7 amino acids in length, said peptide comprising all or part of a tenascin-C region,

said tenascin-C region selected from the group consisting of fnA-D, fnD. and fnC. In a

particular embodiment of this aspect, the peptide comprises the 8-amino acid peptide VFDNFVLK. For example, the peptide is free of any sequence of tenascin-C amino acid seqence

that is both outside the tenascin-C region and exceeds 100 amino acids. In a further related

aspect of the method, the peptide comprises a homologous peptide sequence identical in length

to the tenascin-C region such that, if the homologous amino acid sequence and the tenascin-C

region are aligned and consecutively numbered from the same end. and like numbered amino

acids from the two sequences are compared, there is at least N percent identity between the

amino acids of the homologous sequence and the amino acids of the tenascin-C sequence. N

being 70. N more preferably being 80, N even more preferably being 90. To illustrate the

calculation, consider the following two aligned hypothetical 20-mer peptides:

AAAAAAAAAAVVVVVVVVVV

AAAAAAAGGGGGGVVVVVVV

At the 20 positions along the sequences, there is identity at 14. Therefore N is 70.

In one general aspect the purpose of the methods of the invention is to stimulate axonal

and/or dendritic growth and. in particular embodiments, to stimulate axonal growth independent

of axonal and/or dendritic guidance. Converseh . in another general aspect, the purpose is to

stimulate axonal and/or dendritic guidance and in particular embodiments to stimulate axonal

and/or dendritic guidance independent of axonal and/or dendritic growth.

Any of the methods of the invention can utilize am of the peptides of the invention.

In another aspect, the invention is any DNA molecule coding for any peptide of the invention.

For all the peptides of the invention, a preferred embodiment is one in which the peptide

is a purified peptide or in purified preparation of that peptide. for example, as an isolated peptide.

Synthetically made peptides are examples of such preferred embodiments, as are ones

synthesized in a non-human cell, especialh a non-mammalian cell.

Similarly, for all the DNA molecules of the invention, a preferred embodiment is one in which the DNA molecule is a purified DNA molecule or in a purified preparation of that DNA

molecule, for example as an isolated DNA molecule. Examples of such preferred embodiments

are synthetically made DNA molecules, ones made by recombinant DNA technology, or ones

synthesized by replication in a non-human cell, especially a non-mammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Multidomain structure of human tenascin-C. This diagram is adapted from

Aukhil et al. ( 1993. J. Biol. Chem. 268:2542-2553). The N termini of three arms are joined to

form a trimer, and two trimers are connected via a disulfidc bond to form a hexamer. Each arm

consists of 14 EGF domains. 8- 15 FN-III domains depending on alternative R A splicing, and

a single fibrinogen domain. The universal FN-III domains (fn l -5 and fn6-8) are present in all

tenascin-C splice variants. The largest tenascin-C splice variant contains 7 alternatively spliced

FN-III domains (designated Al , A2, A4, B, C, and D. or fnA-D) which are missing in the shortest

splice variant.

Figure 2. Schematic diagram illustrating the neurite guidance assay. A drop of the

protein of interest in solution was placed in the center of a PLL-coated glass coverslip and

allowed to bind. Excess protein solution was washed away, creating a protein/PLL interface.

Cerebellar granule neurons were cultured for 48 hours on the coverslip. and double

immunocvtochemistry was performed at this time using an antibody against the protein in the

drop and an antibody against neurofilament. Neurite behavior at the interface w as then analyzed

for neurites originating on PLL as well as for neurites originating on the protein spot. The

following criteria were established for the guidance assay: only single, nonfasiculated neurites

within 10 μm of the interface were considered. In addition, only neurites moving toward the

interface were counted, and no neurite whose soma was sitting on the interface was counted. Figure 3. The alternatively spliced region of tenascin-C provides permissive neurite

guidance cues. (A) Cerebellar granule neurons were cultured for 48 hours on PLL-coated

coverslips containing spots of fnl -5. fnA-D, fn6-8, large tenascin-C (TN.L) or small tenascin-C

(TN.S). The percentage of neurites that crossed from PLL to the protein spot and vice versa was

then assessed. Bars represent the mean ± SEM (n= 4). In control experiments, 51 ± 4% of the

neurites crossed from PLL to a fluorescein-labeled BSA control and 50 ± 2% crossed from BSA

to PLL. Neurite behavior at ihl -5 or fn6-8/PLL interfaces did not vary significantly from the

control. The percentage of neurites crossing from PLL to fnA-D was significantly higher than

control (asterisk), and the percentage of neurites crossing from fnA-D to PLL was significantly

lower than control (double asterisk) (p < 0.05; Student-Newman-Keuls test). In contrast to fnA-

D, the percentage of neurites crossing from PLL to large or small tenascin-C (crosses) was

significantly lower than control (p < 0.05; Student-Newman-Keuls test). A polyclonal antibody

(pAb) against fnA-D further reduced the percentage of neurites crossing to large tenascin-C

(double cross); the reduction was significant (p < 0.05; Studcnt-Ncwman-Keuls test). (B ) Double

immunocvtochemistry was performed using a poh clonal antibody against full-length tenascin-C

followed by a fluorescein-coiηugated secondary antibody to detect fnA-D spots, and monoclonal

antibody RT97 followed by a rhodamine-conjugated secondary antibod} to detect neurons.

Neurites on both sides of the PLL/fnA-D interlace showed a preference for fnA-D. Bar, 10 μm.

Figure 4. FnA-D overcomes tenascin-C boundaries to neurite advance. Cerebellar

granule neurons were cultured for 48 hours on PLL-coated coverslips containing spots of protein

comprised of both small tenascin-C and fnA-D (A) or large tenascin-C and fnA-D (B) (n = 3 ).

The concentration of tenascin-C was held constant at 1 00 nM while that of fnA-D was increased

from 100 nM (a 1 : 1 ratio of fnA-D to tenascin-C) to 400 nlVl (a 4: 1 ratio). The percentage of

neurites crossing from PLL to the tenascm-C/fnA-D spot increased with increasing concentrations of fnA-D. The maximal effect was observed with 300 nM fnA-D for small tenascin-C and 200 nM fnA-D for large tenascin-C.

Figure 5. The neurite outgrowth promoting site in fnD does not mediate neurite

guidance. (A) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips containing spots of fnA-D, a mixture of fnA-D and monoclonal antibody (mAb) Jl/tn2, or a mixture of fnA-D and monoclonal antibody tenascin III-B (mAb III-B) (n = 4). Jl/tn2, which

reacts in fnD, did not change the percentage of neurites that crossed from PLL to fnA-D, nor did

mAb III-B, which reacts in fnB. (B) Cerebellar granule neurons were allowed to extend neurites for 48 hours on PLL-coated glass coverslips or PLL-coated glass coverslips to which fnA-D or mixtures of fnA-D and J l/tn2 or mAb III-B had been adsorbed. Distributions of the total neurite length are presented as a box-and-whisker plot. One representative experiment of 4 is shown.

Boxes enclose 25th and 75th percentiles of each distribution and are bisected by the median; whiskers indicate 5th and 95th percentiles. FnA-D facilitated neurite outgrowth in comparison to PLL alone. Jl/tn2 eliminated the neurite outgrowth promoting qualities of fnA-D. whereas mAb III-B had no effect.

Figure 6. Neurite guidance is localized to the C terminal portion of fnA-D. Cerebellar

granule neurons were cultured for 48 hours on PLL-coated coverslips containing spots of fnAl -

A4. fnB-D, or a mixture of fnA 1 -A4 and fiiB-D (n = 3). FnA 1 -A4 formed an inhibitory boundary

to neurites originating on PLL; behavior for neurites originating on fnAl-A4 was more or less random. (The dashed line indicates random neurite behavior at a BSA/PLL interface.) On the other hand, neurites originating on either PLL or fnB-D showed a preference for fnB-D. A

mixture of fnAl-A4 and fnB-D also mimicked the actions of fnA-D; thus fnB-D overcame the

boundary formed by fnAl-A4.

Figure 7. FnC is implicated in mediation of neurite guidance. (A) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips with containing spots of fnA-D or fnA-D (-) C) (n = 3). Neurites on the PLL side of the interface preferred fnA-D but avoided fnA-

D (-) C. Neurites on the protein side of the interface preferred fnA-D but demonstrated random behavior (dashed line) on fnA-D (-) C. (B) Cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips to which fnA-D or fnA-D (-) C had been adsorbed. One representative experiment of 4 is shown. FnA-D and fnA-D (-) C both facilitated neurite outgrowth in comparison to PLL; distributions of neurite length on fnA-D and fnA-D (-) C were

the same.

Figure 8. FnA-D guides neurites in the context of cellular tenascin-C. (A) Cerebellar

granule neurons were cultured for 48 hours on a mixed monolayer of BHK cells and BHK-TN.L or BHK-TN.S cells. Double immunocytochemistry was performed using a polyclonal antibody against full-length tenascin-C followed by a fluorescein-conjugated secondary antibody, and monoclonal antibody RT97 followed by a rhodamine-conjugated secondary antibody. Neurites

crossed from BHK cells to BHK-TN.L cells but avoided BHK-TN.S cells. Bar, 12 μm. (B) Neurite behavior at cellular interfaces was quantified (n = 4). In control experiments. 45-50% of the neurites crossed from BHK cells to PKH26-labeled BHK cells, and vice versa. The

percentage of neurites that crossed from BHK cells to BHK-TN.L cells was significantly higher than control (asterisk), and the percentage that crossed from BHK-TN.L cells to BHK cells was

significantly lower (double asterisk) (p < 0.05; Student-Newman-Keuls test). In contrast, the

percentage of neurites that crossed onto BHK-TN.S cells was significantly lower than control

(cross), and the percentage that crossed off was significantly higher (double cross) (p < 0.05;

Student-Newman-Keuls test).

Figure 9. Neurite guidance in the presence of tenascin-C antibodies. (A) Cerebellar

granule neurons were cultured for 48 hours on a mixed monolayer of BHK cells and BHK-TN.L or BHK-TN.S cells in the presence of a polyclonal antibody against full-length tenascin-C. The

antibody significantly reduced the percentage of neurites that crossed from BHK cells to BHK-

TN.L cells from about 70 to 50% (asterisk) and significantly increased the percentage of neurites

that crossed to BHK-TN.S cells from about 20 to 50% (cross) (p < 0.05; Student-Newman-Keuls

test). (B) Neurons were also cultured on a mixed monolayer of BHK cells and BHK-TN.L cells

in the presence of polyclonal antibodies against fn 1 -5 or fnA-D. or monoclonal antibody (mAb)

Jl /tn2. The fnl -5 antibody did not effect the percentage of neurites that crossed to BHK-TN.L

cells, nor did Jl/tn2. The fnA-D antibody significantly reduced the percentage of neurites that

crossed to about 20% (double asterisk) (/. < 0.05; Student-Newman-Keuls test).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following embodiments and Examples are intended to illustrate not limit the

invention.

Abbreviations

Abbreviations used in this application include: CFDA. carboxy fluorescein diacetate; CNS, central nervous system CSPG. c ondroitin sulfate proteoglycan: EGF. epidermal growth factor; fbg, fibrinogen; FN-I I I. fibronectin type I I I; fn. FN-III domain; mΛb. monoclonal antibody: pAb, polyclonal. TN.L, large tenascin splice variant; TN.S, small tenascin splice variant.

The following one letter codes are used to represent amino acids:

S-scrine. T-threonine. N-asparagine, Q-glutamine. K-lysine. R-arginine.

H-histidine. E-glutamic acid. D-aspartic acid, C-cystine. G-glycine.

P-proline. A-alanine. I-isoleucine. L-leucine. M-methionine. F-phenylalanine, W-tryptophan, V-valine, Y-tyrosine, X-any amino acid.

The following one letter codes are used to represent nucleic acids:

A-adenine, C-cytosine, G-guanine, T-thymidine. R represents A or G, Y

represents T or C, N represents any nucleic acid.

Amino acid sequence of Tenascin-C and DNA coding for it

The following amino acid and nucleic acid sequences are human tenascin-C sequences

from Genbank. Accession Number X78565 Version X78565.1, the nucleic acid sequences including designations LOCUS, HSTENAS3 ; 7560 bp mR A; H.sapiens mRNA for tenascin-C,

7560bp.

/1rans1ation= "MGA TQLLAGVFLAFLALATEGGV KKVIRHKRQSGV ATLPEE NQPWFNHλATNIKLPVGSQCSVDLESASGEKDLAPPSEPSESFQEHTVDGENQIVFTH RINIPRRACGCAAAPDV EL SRLEE ENLVSSLREQCTAGAGCCLQPATGRLDTRPF

CSGRGNFSTEGCGCVCEPG KGPNCSEPECPGNCH RGRCIDGQCICDDGFTGEDCSQ LACPSDCNDQGKCWGVCICFEGYAGADCSREICPVPCSEEHGTCVDGLCVCHDGFAG DDCNKP CLNNCYNRGRCVENECVCDEGFTGEDCSELICPNDCFDRGRCINGTCYCEE GFTGEDCGKPTCPHACHTQGRCEEGQCVCDEGFAG DCSEKRCPADCHNRGRCVDGRC ECDDGFTGADCGELKCPNGCSGHGRCVNGQCVCDEGYTGEDCSQLRCPNDCHSRGRCV

EGKCVCEQGFKGYDCSDMSCPNDCHQHGRCVNGMCVCDDGYTGEDCRDRQCPRDCSNR GLCλ/DGQCVCEDGFTGPDCAELSCPNDCHGQGRCVNGQCVCHEGFMGKDCKEQRCPSD CHGQGRCVDGQCICHEGFTGLDCGQHSCPSDCNN GQCVSGRCICNEGYSGEDCSEVS PPKD WTEVTEETWLAWDNEMRVTEYL YTPTHEGGLEMQFRVPGDQTSTIIQEL EPGVEYFIRVFAILENKKSIPVSARVATYLPAPEGLKFKSIKETSVEVE DPLDIAFE

TWEIIFRNMNKΞDEGEITKSLRRPETSYRQTGLAPGQEYEISLHIVKNNTRGPGLKRV TTTRLDAPSQIEVKDVTDTTALITWFKP AEIDGIELTYGIKDVPGDRTTIDLTEDEN QYSIGNLKPDTEYEVS ISRRGDMSSNPAKETFTTGLDAPRNLRRVSQTDNSIT EWR NGKAAIDSYRIKYAPISGGDHAEVDVPKSQQATTKTTLTG RPGTEYGIGVSAVKEDK ESNPATINAATELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQ VGVQ PRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWD

GLRLN TAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYG VIQGYRTPVLSAEASTGETPN GEVWAEVGWDA KLN TAPEGAYEYFFIQVQEADT VEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTEEVPDMGNL TVTEVSWDALRLNWTTPDGTYDQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGT PYTVT HGEVRGHSTRPLAVEWTEDLPQLGDLAVSEVG DGLRLNWTAADNAYEHFV

IQVQEWKVEAAQNLT PGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPV SAEASTA KEPEIGNLNVSDITPESFNLS MATDGIFETFTIEIIDSNRLLETVEYNISGAERTAH ISGLPPSTDFIλATLSG APSIRTKTISATATTEALPL ΞNLTISDINPYGFTVS MAS ENAFDSFLVTWDSGKLLDPQEFTLSGTQRKLELRG ITGIGYEVMVSGFTQGHQTKP LRAEIVTEAEPEVDNLLVSDATPDGFR SWTADEGVFDNFVLKIRDTKKQSEPLEITL APERTRDLTGLREATEYEIELYGISKGRRSQTVSAlATTAMGSPKEVIFSDITENSA TVS RAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVK IPGVEYLVS11AMKG FEESEPVSGSFTTALDGPSGLVTANITDSEALAR QPAIATVDSYVISYTGEKVPEIT RTVSGNTVEYALTDLEPATEYTLRIFAEKGPQKSSTITAKFTTDLDSPRDLTATEVQS ETALLTWRPPRASVTGYLLVYESVDGTVKEVIVGPDTTSYSLADLSPSTHYTAKIQAL

NGPLRSNMIQTIFTTIGLLYPFPKDCSQAMLNGDTTSG YTIYLNGDKAQALEVFCDM TSDGGG IVFLRRK GRENFYQN KAYAAGFGDRREEF GLDNLNKITAQGQYELRV DLRDHGETAFAVYDKFSVGDAKTRYKLKVEGYΞGTAGDSMAYHNGRSFSTFDKDTDSA ITNCALSYKGAFWYRNCHR-VN MGRYGDNNHSQGVNWFH KGHEHSIQFAEMKLRPSN FRNLEGRRKRA" (SEQ ID NO : 2 ) polyA_signal 7522..7527 ORIGIN

1 accggccaca gcctgcctac tgtcacccgc ctctcccgcg cgcagataca cgcccccgcc 61 tccgtgggca caaaggcagc gctgctgggg aactcggggg aacgcgcacg tgggaaccgc 121 cgcagctcca cactccaggt acttcttcca aggacctagg tctctcgccc atcggaaaga

181 aaataattct ttcaagaaga tcagggacaa ctgatttgaa gtctactctg tgcttctaaa

241 tccccaattc tgctgaaagt gaatccctag agccctagag ccccagcagc acccagccaa

301 acccacctcc accatggggg ccatgactca gctgttggca ggtgtctttc ttgctttcct 361 tgccctcgct accgaaggtg gggtcctcaa gaaagtcatc cggcacaagc gacagagtgg

421 ggtgaacgcc accctgccag aagagaacca gccagtggtg tttaaccacg tttacaacat

481 caagctgcca gtgggatccc agtgttcggt ggatctggag tcagccagtg gggagaaaga

541 cctggcaccg ccttcagagc ccagcgaaag ctttcaggag cacacagtag atggggaaaa

601 ccagattgtc ttcacacatc gcatcaacat cccccgccgg gcctgtggct gtgccgcagc 661 ccctgatgtt aaggagctgc tgagcagact ggaggagctg gagaacctgg tgtcttccct

721 gagggagcaa tgtactgcag gagcaggctg ctgtctccag cctgccacag gccgcttgga

781 caccaggccc ttctgtagcg gtcggggcaa cttcagcact gaaggatgtg gctgtgtctg

841 cgaacctggc tggaaaggcc ccaactgctc tgagcccgaa tgtccaggca actgtcacct

901 tcgaggccgg tgcattgatg ggcagtgcat ctgtgacgac ggcttcacgg gcgaggactg 961 cagccagctg gcttgcccca gcgactgcaa tgaccagggc aagtgcgtga atggagtctg

1021 catctgtttc gaaggctacg ccggggctga ctgcagccgt gaaatctgcc cagtgccctg

1081 cagtgaggag cacggcacat gtgtagatgg cttgtgtgtg tgccacgatg gctttgcagg

1141 cgatgactgc aacaagcctc tgtgtctcaa caattgctac aaccgtggac gatgcgtgga

1201 gaatgagtgc gtgtgtgatg agggtttcac gggcgaagac t agtgagc tcatctgccc 1261 caatgactgc ttcgaccggg gccgctgcat caatggcacc tgctactgcg aagaaggctt

1321 cacaggtgaa gactgcggga aacccacctg cccacatgcc tgccacaccc agggccggtg

1381 tgaggagggg cagtgtgtat gtgatgaggg ctttgccggt ttggactgca gcgagaagag

1441 gtgtcctgct gactgtcaca atcgtggccg ctgtgtagac gggcggtgtg agtgtgatga

1501 tggtttcact ggagctgact gtggggagct caagtgtccc aatggctgca gtggccatgg 1561 ccgctgtgtc aatgggcagt gtgtgtgtga tgagggctat actggggagg actgcagcca

1621 gctacggtgc cccaatgact gtcacagtcg gggccgctgt gtcgagggca aatgtgtatg

1681 tgagcaaggc ttcaagggct atgactgcag tgacatgagc tgccctaatg actgtcacca

1741 gcacggccgc tgtgtgaatg gcatgtgtgt ttgtgatgac ggctacacag gggaagactg

1801 ccgggatcgc caatgcccca gggactgcag caacaggggc ctctgtgtgg acggacagtg 1861 cgtctgtgag gacggcttca ccggccctga ctgtgcagaa ctctcctgtc caaatgactg 1921 ccatggccag ggtcgctgtg tgaatgggca gtgcgtgtgc catgaaggat ttatgggcaa 1981 agactgcaag gagcaaagat gtcccagtga ctgtcatggc cagggccgct gcgtggacgg 2041 ccagtgcatc tgccacgagg gcttcacagg cctggactgt ggccagcact cctgccccag 2101 tgactgcaac aacttaggac aatgcgtctc gggccgctgc atctgcaacg agggctacag

2161 cggagaagac tgctcagagg tgtctcctcc caaagacctc gttgtgacag aagtgacgga 2221 agagacggtc aacctggcct gggacaatga gatgcgggtc acagagtacc ttgtcgtgta 2281 cacgcccacc cacgagggtg gtctggaaat gcagttccgt gtgcctgggg accagacgtc 2341 caccatcatc caggagctgg agcctggtgt ggagtacttt atccgtgtat ttgccatcct 2401 ggagaacaag aagagcattc ctgtcagcgc cagggtggcc acgtacttac ctgcacctga

2461 aggcctgaaa ttcaagtcca tcaaggagac atctgtggaa gtggagtggg atcctctaga 2521 cattgctttt gaaacctggg agatcatctt ccggaatatg aataaagaag atgagggaga 2581 gatcaccaaa agcctgagga ggccagagac ctcttaccgg caaactggtc tagctcctgg 2641 gcaagagtat gagatatctc tgcacatagt gaaaaacaat acccggggcc ctggcctgaa 2701 gagggtgacc accacacgct tggatgcccc cagccagatc gaggtgaaag atgtcacaga

2761 caccactgcc ttgatcacct ggttcaagcc cctggctgag atcgatggca ttgagctgac 2821 ctacggcatc aaagacgtgc caggagaccg taccaccatc gatctcacag aggacgagaa 2881 ccagtactcc atcgggaacc tgaagcctga cactgagtac gaggtgtccc tcatctcccg 2941 cagaggtgac atgtcaagca acccagccaa agagaccttc acaacaggcc tcgatgctcc 3001 caggaatctt cgacgtgttt cccagacaga taacagcatc accctggaat ggaggaatgg

3061 caaggcagct attgacagtt acagaattaa gtatgccccc atctctggag gggaccacgc 3121 tgaggttgat gttccaaaga gccaacaagc cacaaccaaa accacactca caggtctgag 3181 gccgggaact gaatatggga ttggagtttc tgctgtgaag gaagacaagg agagcaatcc 3241 agcgaccatc aacgcagcca cagagttgga cacgcccaag gaccttcagg tttctgaaac 3301 tgcagagacc agcctgaccc tgctctggaa gacaccgttg gccaaatttg accgctaccg

3361 cctcaattac agtctcccca caggccagtg ggtgggagtg cagcttccaa gaaacaccac 3421 ttcctatgtc ctgagaggcc tggaaccagg acaggagtac aatgtcctcc tgacagccga 3481 gaaaggcaga cacaagagca agcccgcacg tgtgaaggca tccactgaac aagcccctga 3541 gctggaaaac ctcaccgtga ctgaggttgg ctgggatggc ctcagactca actggaccgc 3601 ggctgaccag gcctatgagc actttatcat tcaggtgcag gaggccaaca aggtggaggc

3661 agctcggaac ctcaccgtgc ctggcagcct tcgggctgtg gacataccgg gcctcaaggc

3721 tgctacgcct tatacagtct ccatctatgg ggtgatccag ggctatagaa caccagtgct

3781 ctctgctgag gcctccacag gggaaactcc caatttggga gaggtcgtgg tggccgaggt 3841 gggctgggat gccctcaaac tcaactggac tgctccagaa ggggcctatg agtacttttt

3901 cattcaggtg caggaggctg acacagtaga ggcagcccag aacctcaccg tcccaggagg

3961 actgaggtcc acagacctgc ctgggctcaa agcagccact cattatacca tcaccatccg

4021 cggggtcact caggacttca gcacaacccc tctctctgtt gaagtcttga cagaggaggt

4081 tccagatatg ggaaacctca cagtgaccga ggttagctgg gatgctctca gactgaactg 4141 gaccacgcca gatggaacct atgaccagtt tactattcag gtccaggagg ctgaccaggt

4201 ggaagaggct cacaatctca cggttcctgg cagcctgcgt tccatggaaa tcccaggcct

4261 cagggctggc actccttaca cagtcaccct gcacggcgag gtcaggggcc acagcactcg

4321 accccttgct gtagaggtcg tcacagagga tctcccacag ctgggagatt tagccgtgtc

4381 tgaggttggc tgggatggcc tcagactcaa ctggaccgca gctgacaatg cctatgagca 4441 ctttgtcatt caggtgcagg aggtcaacaa agtggaggca gcccagaacc tcacgttgcc

4501 tggcagcctc agggctgtgg acatcccggg cctcgaggct gccacgcctt atagagtctc

4561 catctatggg gtgatccggg gctatagaac accagtactc tctgctgagg cctccacagc

4621 caaagaacct gaaattggaa acttaaatgt ttctgacata actcccgaga gcttcaatct

4681 ctcctggatg gctaccgatg ggatcttcga gacctttacc attgaaatta ttgattccaa 4741 taggttgctg gagactgtgg aatataatat ctctggtgct gaacgaactg cccatatctc

4801 agggctaccc cctagtactg attttattgt ctacctctct ggacttgctc ccagcatccg

4861 gaccaaaacc atcagtgcca cagccacgac agaggccctg ccccttctgg aaaacctaac

4921 catttccgac attaatccct acgggttcac agtttcctgg atggcatcgg agaatgcctt

4981 tgacagcttt ctagtaacgg tggtggattc tgggaagctg ctggaccccc aggaattcac 5041 actttcagga acccagagga agctggagct tagaggcctc ataactggca ttggctatga

5101 ggttatggtc tctggcttca cccaagggca tcaaaccaag cccttgaggg ctgagattgt

5161 tacagaagcc gaaccggaag ttgacaacct tctggtttca gatgccaccc cagacggttt

5221 ccgtctgtcc tggacagctg atgaaggggt cttcgacaat tttgttctca aaatcagaga

5281 taccaaaaag cagtctgagc cactggaaat aaccctactt gcccccgaac gtaccaggga 5341 cttaacaggt ctcagagagg ctactgaata cgaaattgaa ctctatggaa taagcaaagg 5401 aaggcgatcc cagacagtca gtgctatagc aacaacagcc atgggctccc caaaggaagt 5461 cattttctca gacatcactg aaaattcggc tactgtcagc tggagggcac ccacggccca 5521 agtggagagc ttccggatta cctatgtgcc cattacagga ggtacaccct ccatggtaac 5581 tgtggacgga accaagactc agaccaggct ggtgaaactc atacctggcg tggagtacct

5641 tgtcagcatc atcgccatga agggctttga ggaaagtgaa cctgtctcag ggtcattcac 5701 cacagctctg gatggcccat ctggcctggt gacagccaac atcactgact cagaagcctt 5761 ggccaggtgg cagccagcca ttgccactgt ggacagttat gtcatctcct acacaggcga 5821 gaaagtgcca gaaattacac gcacggtgtc cgggaacaca gtggagtatg ctctgaccga 5881 cctcgagcct gccacggaat acacactgag aatctttgca gagaaagggc cccagaagag

5941 ctcaaccatc actgccaagt tcacaacaga cctcgattct ccaagagact tgactgctac 6001 tgaggttcag tcggaaactg ccctccttac ctggcgaccc ccccgggcat cagtcaccgg 6061 ttacctgctg gtctatgaat cagtggatgg cacagtcaag gaagtcattg tgggtccaga 6121 taccacctcc tacagcctgg cagacctgag cccatccacc cactacacag ccaagatcca 6181 ggcactcaat gggcccctga ggagcaatat gatccagacc atcttcacca caattggact

6241 cctgtacccc ttccccaagg actgctccca agcaatgctg aatggagaca cgacctctgg 6301 cctctacacc atttatctga atggtgataa ggctcaggcg ctggaagtct tctgtgacat 6361 gacctctgat gggggtggat ggattgtgtt cctgagacgc aaaaacggac gcgagaactt 6421 ctaccaaaac tggaaggcat atgctgctgg atttggggac cgcagagaag aattctggct 6481 tgggctggac aacctgaaca aaatcacagc ccaggggcag tacgagctcc gggtggacct

6541 gcgggaccat ggggagacag cctttgctgt ctatgacaag ttcagcgtgg gagatgccaa 6601 gactcgctac aagctgaagg tggaggggta cagtgggaca gcaggtgact ccatggccta 6661 ccacaatggc agatccttct ccacctttga caaggacaca gattcagcca tcaccaactg 6721 tgctctgtcc tacaaagggg ctttctggta caggaactgt caccgtgtca acctgatggg 6781 gagatatggg gacaataacc acagtcaggg cgttaactgg ttccactgga agggccacga

6841 acactcaatc cagtttgctg agatgaagct gagaccaagc aacttcagaa atcttgaagg 6901 caggcgcaaa cgggcataaa ttggagggac cactgggtga gagaggaata aggcggccca 6961 gagcgaggaa aggattttac caaagcatca atacaaccag cccaaccatc ggtccacacc 7021 tgggcatttg gtgagaatca aagctgacca tggatccctg gggccaacgg caacagcatg 7081 ggcctcacct cctctgtgat ttctttcttt gcaccaaaga catcagtctc caacatgttt 7141 ctgttttgtt gtttgattca gcaaaaatct cccagtgaca acatcgcaat agttttttac 7201 ttctcttagg tggctctggg atgggagagg ggtaggatgt acaggggtag tttgttttag 7261 aaccagccgt attttacatg aagctgtata attaattgtc attatttttg ttagcaaaga 7321 ttaaatgtgt cattggaagc catccctttt tttacatttc atacaacaga aaccagaaaa

7381 gcaatactgt ttccatttta aggatatgat taatattatt aatataataa tgatgatgat 7441 gatgatgaaa actaaggatt tttcaagaga tctttctttc caaaacattt ctggacagta 7501 cctgattgta tttttttttt aaataaaagc acaagtactt ttgaaaaaaa accggaattc

//(SEQIDNO:3)

Subregions of Tenascin-C the peptide VFDNFVLK is amino acids 1646-1653 (SEQ ID NO:l);

fnA-D is amino acids 1072-1078 (SEQ ID NO:8); fnA-D being alternatively spliced FN-III domain D is amino acidslόl 8-1708 (SEQ ID

NO:9); fnA-D being alternatively spliced domain C is amino acids 1527-1617 (Seq ID NO: 10); fnD is another term for alternatively spliced FN-III domain D; and

fnC is another term for alternatively spliced FN-III domain C.

Integrin alpha 7 precursor sequence

This amino acid sequence and related information and summary was obtained through

the NCBI Entrez protein database.

LOCUS NP 002197 1 137 aa PRI 01 -OCT-1999 WO 00/6662 PCT/USOO/l 1647

DEFINITION integrin alpha 7 precursor [Homo sapiens].

ACCESSION NP_002197

VERSION NP 002197.1 GI.4504753

ITGA7 encodes integrin alpha chain 7. Integrins are heterodimeric integral membrane proteins

composed of an alpha chain and a beta chain. Alpha chain 7 undergoes post-translational

cleavage within the extracellular domain to yield disulfide-linked light and heavy chains that join

with beta 1 to form an integrin that binds to the extracellular matrix protein laminin- 1 .

ORIGIN

1 magarsrdpw gasgicylfg sllvellfsr avafnldvmg alrkegepgs lfgfsvalhr 61 qlqprpqswl lvgapqalal pgqqanrtgg lfacplslee tdcyrvdidq gadmqkeske 121 nq lgvsvrs qgpggkivtc ahryearqrv dqiletrdmi grcfvlsqdl airdeldgge 181 wkfcegrpqg heqfgfcqqg taaafspdsh yllfgapgty nwkgllfvtn idssdpdqlv 241 yktldpadrl pgpagdlaln sylgfsidsg kglvraeels fvagapranh kgavvilrkd 301 sasrlvpevm lsgerltsgf gyslavadln sdgwpdlivg apyfferqee lggavyvyln 361 qgghwagisp lrlcgspdsm fgislavlgd lnqdgfpdia vgapfdgdgk vfiyhgsslg 421 vvakpsqvle geavgiksfg yslsgsldmd gnqypdllvg sladtavlfr arpilhvshe 481 vsiaprsidl eqpncagghs vcvdlrvcfs yiavpssysp tvaldyvlda dtdrrlrgqv 541 prvtflsrnl eepkhqasgt vwlkhqhdrv cgdamfqlqe nvkdklraiv vtlsyslqtp 601 rlrrqapgqg lppvapilna hqpstqraei hflkqgcged kicqsnlqlv harfctrvsd 661 tefqplpmdv dgttalfals gqpviglelm vtnlpsdpaq pqadgddahe aqllvmlpds 721 lhysgvrald paekplclsn enashvecel gnpmkrgaqv tfylilstsg isiettelev 781 elllatiseq elhpvsarar vfielplsia gmaipqqlff sgvvrgeram qserdvgskv WO 00/66628 PCT/USOO/l 1647

841 kyevtvsnqg qslrtlgsaf lnimwpheia ngk llypmq veleggqgpg qkglcsprpn

901 ilhldvdsrd rrrreleppe qqepgerqep smswwpvssa ekkknitldc argtancvvf

961 scplysfdra avlhvwgrlw nstfleeysa vkslevivra nitvkssikn lmlrdastvi

1021 pvmvyldpma vvaegvpwwv illavlagll vlallvlllw kmgffkrakh peatvpqyha 1081 vkipredrqq fkeektgtil rnnwgsprre gpdahpilaa dghpelgpdg hpgpgta

// (SEQ ID NO:4)

This nucleotide sequence and related information and summary was obtained through

the NCBI Entrez nucleotide database. LOCUS NM_002206 4079 bp mRNA PRI 01-OCT-

1999

DEFINITION Homo sapiens integrin, alpha 7 (ITGA7) mRNA.

ACCESSION NM_002206

VERSION NM_002206.1 GL4504752 ORIGIN

1 ggagcggcgg gcgggcggga gggctggcgg ggcgaacgtc tgggagacgt ctgaaagacc

61 aacgagactt tggagaccag agacgcgcct ggggggacct ggggcttggg gcgtgcgaga

121 tttcccttgc attcgctggg agctcgcgca gggatcgtcc catggccggg gctcggagcc

181 gcgacccttg gggggcctcc gggatttgct acctttttgg ctccctgctc gtcgaactgc 241 tcttctcacg ggctgtcgcc ttcaatctgg acgtgatggg tgccttgcgc aaggagggcg

301 agccaggcag cctcttcggc ttctctgtgg ccctgcaccg gcagttgcag ccccgacccc 361 agagctggct gctggtgggt gctccccagg ccctggctct tcctgggcag caggcgaatc 421 gcactggagg cctcttcgct tgcccgttga gcctggagga gactgactgc tacagagtgg

481 acatcgacca gggagctgat atgcaaaagg aaagcaagga gaaccagtgg ttgggagtca 541 gtgttcggag ccaggggcct gggggcaaga ttgttacctg tgcacaccga tatgaggcaa

601 ggcagcgagt ggaccagatc ctggagacgc gggatatgat tggtcgctgc tttgtgctca 661 gccaggacct ggccatccgg gatgagttgg atggtgggga atggaagttc tgtgagggac 721 gcccccaagg ccatgaacaa tttgggttct gccagcaggg cacagctgcc gccttctccc 781 ctgatagcca ctacctcctc tttggggccc caggaaccta taattggaag gggttgcttt 841 ttgtgaccaa cattgatagc tcagaccccg accagctggt gtataaaact ttggaccctg

901 ctgaccggct cccaggacca gccggagact tggccctcaa tagctactta ggcttctcta

961 ttgactcggg gaaaggtctg gtgcgtgcag aagagctgag ctttgtggct ggagcccccc

1021 gcgccaacca caagggtgct gtggttatcc tgcgcaagga cagcgccagt cgcctggtgc

1081 ccgaggttat gctgtctggg gagcgcctga cctccggctt tggctactca ctggctgtgg 1141 ctgacctcaa cagtgatggc tggccagacc tgatagtggg tgccccctac ttctttgagc

1201 gccaagaaga gctggggggt gctgtgtatg tgtacttgaa ccaggggggt cactgggctg 1261 ggatctcccc tctccggctc tgcggctccc ctgactccat gttcgggatc agcctggctg 1321 tcctggggga cctcaaccaa gatggctttc cagatattgc agtgggtgcc ccctttgatg

1381 gtgatgggaa agtcttcatc taccatggga gcagcctggg ggttgtcgcc aaaccttcac 1441 aggtgctgga gggcgaggct gtgggcatca agagcttcgg ctactccctg tcaggcagct

1501 tggatatgga tgggaaccaa taccctgacc tgctggtggg ctccctggct gacaccgcag 1561 tgctcttcag ggccagaccc atcctccatg tctcccatga ggtctctatt gctccacgaa 1621 gcatcgacct ggagcagccc aactgtgctg gcggccactc ggtctgtgtg gacctaaggg 1681 tctgtttcag ctacattgca gtccccagca gctatagccc tactgtggcc ctggactatg 1741 tgttagatgc ggacacagac cggaggctcc ggggccaggt tccccgtgtg acgttcctga 1801 gccgtaacct ggaagaaccc aagcaccagg cctcgggcac cgtgtggctg aagcaccagc 1861 atgaccgagt ctgtggagac gccatgttcc agctccagga aaatgtcaaa gacaagcttc 1921 gggccattgt agtgaccttg tcctacagtc tccagacccc tcggctccgg cgacaggctc

1981 ctggccaggg gctgcctcca gtggccccca tcctcaatgc ccaccagccc agcacccagc 2041 gggcagagat ccacttcctg aagcaaggct gtggtgaaga caagatctgc cagagcaatc 2101 tgcagctggt ccacgcccgc ttctgtaccc gggtcagcga cacggaattc caacctctgc 2161 ccatggatgt ggatggaaca acagccctgt ttgcactgag tgggcagcca gtcattggcc 2221 tggagctgat ggtcaccaac ctgccatcgg acccagccca gccccaggct gatggggatg

2281 atgcccatga agcccagctc ctggtcatgc ttcctgactc actgcactac tcaggggtcc 2341 gggccctgga ccctgcggag aagccactct gcctgtccaa tgagaatgcc tcccatgttg 2401 agtgtgagct ggggaacccc atgaagagag gtgcccaggt caccttctac ctcatcctta 2461 gcacctccgg gatcagcatt gagaccacgg aactggaggt agagctgctg ttggccacga 2521 tcagtgagca ggagctgcat ccagtctctg cacgagcccg tgtcttcatt gagctgccac

2581 tgtccattgc aggaatggcc attccccagc aactcttctt ctctggtgtg gtgaggggcg 2641 agagagccat gcagtctgag cgggatgtgg gcagcaaggt caagtatgag gtcacggttt 2701 ccaaccaagg ccagtcgctc agaaccctgg gctctgcctt cctcaacatc atgtggcctc 2761 atgagattgc caatgggaag tggttgctgt acccaatgca ggttgagctg gagggcgggc 2821 aggggcctgg gcagaaaggg ctttgctctc ccaggcccaa catcctccac ctggatgtgg

2881 acagtaggga taggaggcgg cgggagctgg agccacctga gcagcaggag cctggtgagc 2941 ggcaggagcc cagcatgtcc tggtggccag tgtcctctgc tgagaagaag aaaaacatca 3001 ccctggactg cgcccggggc acggccaact gtgtggtgtt cagctgccca ctctacagct 3061 ttgaccgcgc ggctgtgctg catgtctggg gccgtctctg gaacagcacc tttctggagg 3121 agtactcagc tgtgaagtcc ctggaagtga ttgtccgggc caacatcaca gtgaagtcct

3181 ccataaagaa cttgatgctc cgagatgcct ccacagtgat cccagtgatg gtatacttgg 3241 accccatggc tgtggtggca gaaggagtgc cctggtgggt catcctcctg gctgtactgg 3301 ctgggctgct ggtgctagca ctgctggtgc tgctcctgtg gaagatggga ttcttcaaac 3361 gggcgaagca ccccgaggcc accgtgcccc agtaccatgc ggtgaagatt cctcgggaag 3421 accgacagca gttcaaggag gagaagacgg gcaccatcct gaggaacaac tggggcagcc

3481 cccggcggga gggcccggat gcacacccca tcctggctgc tgacgggcat cccgagctgg 3541 gccccgatgg gcatccaggg ccaggcaccg cctaggttcc catgtcccag cctggcctgt 3601 ggctgccctc catcccttcc ccagagatgg ctccttggga tgaagagggt agagtgggct 3661 gctggtgtcg catcaagatt tggcaggatc ggcttcctca ggggcacaga cctctcccac 3721 ccacaagaac tcctcccacc caacttcccc ttagagtgct gtgagatgag agtgggtaaa

3781 tcagggacag ggccatgggg tagggtgaga agggcagggg tgtcctgatg caaaggtggg 3841 gagaagggat cctaatccct tcctctccca ttcaccctgt gtaacaggac cccaaggacc 3901 tgcctccccg gaagtgcctt aacctagagg gtcggggagg aggttgtgtc actgactcag 3961 gctgctcctt ctctagtttc ccctctcatc tgaccttagt ttgctgccat cagtctagtg 4021 gtttcgtggt ttcgtctatt tattaaaaaa tatttgagaa caaaaaaaaa aaaaaaaaa

//

(SEQIDNO:5)

Integrin beta 1 sequence

This amino acid sequence and related information and summary was obtained through the NCBI Entrez nucleotide database.

LOCUS HSFNRB 3614 bp mRNA PRI 12-APR-1999 DEFINITION Human mRNA for integrin beta 1 subunit.

ACCESSION X07979 WO 00/66628 PCT/USOO/l 1647

VERSION X07979.1 GI-31441

/translation="MNLQPIF IGLISSVCCVFAQTDENRCLKANAKSCGECIQAGPN

CG CTNSTFLQEGMPTSARCDDLEALKKKGCPPDDIENPRGSKDIKKNKNVTNRSKGT

AEKLKPEDIHQIQPQQLVLRLRSGEPQTFTLKFKRAEDYPIDLYYLMDLSYSMKDDLE

NVKSLGTDLMNEMRRITSDFRIGFGSFVEKTV PYISTTPAKLRNPCTSEQNCTTPFS

YKNVLSLTNKGEVFNELVGKQRISGNLDSPEGGFDAIMQVAVCGSLIG RNVTRLLVF

STDAGFHFAGDGKLGGIVLPNDGQCHLENNMYT SHYYDYPSIAHLVQKLSENNIQTI

FAVTEEFQPVYKELKNLIPKSAVGTLSANSSNVIQLIIDAYNSLSSEVILENGKLSEG

VTISYKSYCKNGVNGTGENGRKCSNISIGDEVQFEISITSNKCPKKDSDSFKIRPLGF

TEEVEVILQYICECECQSEGIPESPKCHEGNGTFECGACRCNEGRVGRHCECSTDEVN

SEDMDAYCRKENSSEICSNNGECVCGQCVCRKRDNTNEIYSGKFCECDNFNCDRSNGL

ICGGNGVCKCRVCECNPNYTGSACDCSLDTSTCEASNGQICNGRGICECGVCKCTDPK

FQGQTCEMCQTCLGVCAEHKECVQCRAFNKGEKKDTCTQECSYFNITKVESRDKLPQP

VQPDPVSHCKEKDVDDCWFYFTYSVNGNNEVMVHVVENPECPTGPDIIPIVAGVVAGI

VLIGLALLLI KLLMIIHDRREFAKFEKEKMNAK DTGENPIYKSAVTTVVNPKYEGK

(SEQ ID NO:6)

ORIGIN

1 gtccgccaaa acctgcgcgg atagggaaga acagcacccc ggcgccgatt gccgtaccaa

61 acaagcctaa cgtccgctgg gccccggacg ccgcgcggaa aagatgaatt tacaaccaat 121 tttctggatt ggactgatca gttcagtttg ctgtgtgttt gctcaaacag atgaaaatag

181 atgtttaaaa gcaaatgcca aatcatgtgg agaatgtata caagcagggc caaattgtgg

241 gtggtgcaca aattcaacat ttttacagga aggaatgcct acttctgcac gatgtgatga

301 tttagaagcc ttaaaaaaga agggttgccc tccagatgac atagaaaatc ccagaggctc

361 caaagatata aagaaaaata aaaatgtaac caaccgtagc aaaggaacag cagagaagct 421 caagccagag gatattcatc agatccaacc acagcagttg gttttgcgat taagatcagg

481 ggagccacag acatttacat taaaattcaa gagagctqaa gactatccca ttgacctcta

541 ctaccttatg gacctgtctt attcaatgaa agacgatttg gagaatgtaa aaagtcttgg

601 aacagatctg atgaatgaaa tgaggagqat tacttcggac ttcagaattg gatttggctc

661 atttgtggaa aagactgtga tgccttacat tagcacaaca ccagctaagc tcaggaaccc 721 ttgcacaagt gaacagaact gcaccacccc atttagctac aaaaatgtgc tcagtcttac 781 taataaagga gaagtattta atgaacttgt tggaaaacag cgcatatctg gaaatttgga

841 ttctccagaa ggtggtttcg atgccatcat gcaagttgca gtttgtggat cactgattgg

901 ctggaggaat gttacacggc tgctggtgtt ttccacagat gccgggtttc actttgctgg

961 agatgggaaa cttggtggca ttgttttacc aaatgatgga caatgtcacc tggaaaataa 1021 tatgtacaca atgagccatt attatgatta tccttctatt gctcaccttg tccagaaact

1081 gagtgaaaat aatattcaga caatttttgc agttactgaa gaatttcagc ctgtttacaa

1141 ggagctgaaa aacttgatcc ctaagtcagc agtaggaaca ttatctgcaa attctagcaa

1201 tgtaattcag ttgatcattg atgcatacaa ttccctttcc tcagaagtca ttttggaaaa

1261 cggcaaattg tcagaaggag taacaataag ttacaaatct tactgcaaga acggggtgaa 1321 tggaacaggg gaaaatggaa gaaaatgttc caatatttcc attggagatg aggttcaatt 1381 tgaaattagc ataacttcaa ataagtgtcc aaaaaaggat tctgacagct ttaaaattag 1441 gcctctgggc tttacggagg aagtagaggt tattcttcag tacatctgtg aatgtgaatg 1501 ccaaagcgaa ggcatccctg aaagtcccaa gtgtcatqaa ggaaatggga catttgagtg 1561 tggcgcgtgc aggtgcaatg aagggcgtgt tggtagacat tgtgaatgca gcacagatga 1621 agttaacagt gaagacatgg atgcttactq caggaaaαaa aacagttcag aaatctgcag 1681 taacaatgga gagtgcgtct gcggacagtg tgtttgtaqg aagagggata atacaaatga 1741 aatttattct ggcaaattct gcgagtgtga taatttcaac tgtgatagat ccaatggctt 1801 aatttgtgga ggaaatggtg tttgcaagtg tcgtgtgtgt gagtgcaacc ccaactacac 1861 tggcagtgca tgtgactgtt ctttggatac tagtacttgt gaagccagca acggacagat 1921 ctgcaatggc cggggcatct gcgagtgtgg tgtctgtaag tgtacagatc cgaagtttca 1981 agggcaaacg tgtgagatgt gtcagacctg ccttggtqtc tgtgctgagc ataaagaatg 2041 tgttcagtgc agagccttca ataaaggaga aaagaaaqac acatgcacac aggaatgttc 2101 ctattttaac attaccaagg tagaaagtcg ggacaaatta ccccagccgg tccaacctga 2161 tcctgtgtcc cattgtaagg agaaggatgt tgacgactgt tggttctatt ttacgtattc 2221 agtgaatggg aacaacgagg tcatggttca tgttgtgqag aatccagagt gtcccactgg 2281 tccagacatc attccaattg tagctggtgt ggttgctqga attgttctta ttggccttgc 2341 attactgctg atatggaagc ttttaatgat aattcatq.c agaagggagt ttgctaaatt 2401 tgaaaaggag aaaatgaatg ccaaatggga cacgggtqaa aatcctattt ataagagtgc 2461 cgtaacaact gtggtcaatc cgaagtatga gggaaaatqa gtactgcccg tgcaaatccc 2521 acaacactga atgcaaagta gcaatttcca tagtcacaqt taggtagctt tagggcaata 2581 ttgccatggt tttactcatg tgcaggtttt gaaaatgtac aatatgtata atttttaaaa 2641 tgttttatta ttttgaaaat aatgttgtaa ttcatgccag ggactgacaa aagacttgag 2701 acaggatggt tattcttgtc agctaaggtc acattgtgcc tttttgacct tttcttcctg 2761 gactattgaa atcaaqctta ttggattaag tgatatttct atagcgattg aaagggcaat 2821 agttaaagta atgagcatga tgagagtttc tgttaatcat gtattaaaac tgatttttag 2881 ctttacatat gtcagtttgc agttatgcag aatccaaaαt aaatqtcctg ctagctagtt 2941 aaggattgtt ttaaatctgt tattttgcta tttgcctgtt agacatgact gatgacatat 3001 ctgaaagaca agtatgttga gagttgctgg tgtaaaatac gtttgaaata gttgatctac 3061 aaaggccatg ggaaaaattc agagagttag gaaggaaaaa ccaatagctt taaaacctgt 3121 gtgccatttt aagagttact taatgtttgg taacttttat gccttcactt tacaaattca 3181 agccttagat aaaagaaccg agcaattttc tgctaaaaag tccttgattt agcactattt 3241 acatacaggc catactttac aaagtatttg ctgaatgqgg accttttgag ttgaatttat 3301 tttattattt ttattttgtt taatgtctgq tgctttctat cacctcttct aatcttttaa 3361 tgtatttgtt tgcaatttrg gggtaagact tttttatαag tactttttct ttgaagtttt WO 00/66628 PCT/USOO/l1647

3421 agcggtcaat ttgccttttt aatgaacatg tgaagttata ctgtggctat gcaacagctc 3481 tcacctacgc gagtcttact ttgagttagt gccataacag accactgtat gtttacttct 3541 caccatttga gttgcccatc ttgtttcaca ctagtcacat tcttgtttta agtgccttta 3601 gttttaacag ttca // (SEQ ID N0:7)

?? EXAMPLES Example 1

The purpose of this study was to investigate whether fnA-D also provides neurite

guidance cues and whether the same or different sequences mediate outgrowth and guidance.

We developed an assay to quantify neurite behavior at sharp substrate boundaries and found that

neurites demonstrated a strong preference for fnA-D when given a choice at a poly-L-lysine/fnA-

D interface. Furthermore, neurites preferred cells that over expressed the largest but not the

smallest tenascin-C splice variant when given a choice between control cells and cells transfected

with tenascin-C. The permissive guidance cues of large tenascin-C expressed by cells were

mapped to fnA-D. Using a combination of bacterial expression proteins corresponding to

specific alternatively spliced FN-III domains and monoclonal antibodies against neurite

outgrowth promoting sites, we demonstrated that neurite outgrowth and guidance were facilitated

by distinct sequences within fnA-D. Hence neurite outgrowth and neurite guidance mediated by

the alternatively spliced region of tenascin-C are separable events which can be independently

regulated.

Our previous work suggests that neurite outgrowth and guidance may be separable events

(Powell and Geller, 1999). We therefore used two complementary choice assays to investigate

the hypothesis that fnA-D imparts distinct outgrowth and guidance cues. In one. growing

neurites were allowed to choose between poly-L-lysinc and purified expression proteins

corresponding to alternatively spliced and universal tenascin-C FN-III domains, as well as large

and small tenascin-C splice variants. The other assay was designed to investigate the actions of

fnA-D in cellular tenascin-C. which more closely approximates the in vivo environment. Neurites were allowed to choose between transfected cells that over expressed either large or

small tenascin-C in heterogenous monolayers with untransfected cells. Neurites demonstrated

a strong preference for fnA-D which was masked in large tenascin-C on inert substrates and only

revealed in large tenascin-C on cellular substrates (Meiners and Geller, 1997: Meiners et al.,

1999). Guidance and outgrowth promotion were further localized to different sequences within

fnA-D using monoclonal antibodies against neurite outgrowth promoting sites and expression

proteins coπ-esponding to specific alternatively spliced FN-III domains. Hence neurite outgrowth

and guidance can be independently regulated by the alternatively spliced region of tenascin-C.

Materials and Methods

Proteins and Antibodies

Transfected baby hamster kidney (BHK) cells, bacterial expression proteins, and rabbit

polyclonal tenascin-C antibodies were gifts of Dr. Harold Erickson (Department of Cell Biology,

Duke University Medical Center. Durham, NC). Splice variants of human tenascin-C were

produced in the transfected cells (Aukhil et al.. 1 93). Native large and small tenascin-C were

purified from culture supernatants of these cells by gelatin-sepharos and hydroxyapatite

chromatography (Aukhil et al., 1990; Erickson and Briscoc. 1995) followed by electroelution

from nondenaturing gels (Ho. S.-Y.. Palnitkar. S.. and Meiners. S., unpublished data). Bacterial

expression proteins (Aukhil et al.. 1993) corresponded to universal FN-III domains 1-5 and 6-8

(fnl -5 and fn6-8), fnA-D, the alternatively spliced FN-III domains of large tenascin-C; and fnA-

D (-) C, the alternatively spliced domains minus FN-III domain C (fnC). Fnl-5. fn6-8, and fnA-

D were produced using the polymerase chain reaction (PCR) and cDNA isolated from BHK cells

transfected with large tenascin-C as the template. FnA-D (-) C was produced using PCR and

cDNA isolated from U251 -MG glioma cells as the template. (U251-MG cells produce alternatively spliced transcripts of tenascin-C that contain fnA-D as well as fnA-D (-) C, although

the species that contains fnA-D predominates (Erickson and Bourdon, 1989).) Rabbit polyclonal

full-length tenascin-C antibody was prepared against highly purified tenascin-C from U251 -MG

cells, which is almost entirely large tenascin-C (Erickson and Bourdon, 1989). Rabbit polyclonal

antibodies against fnl-5 and fnA-D were prepared against the corresponding expression protein.

All reagents cited correspond to the human protein.

Bacterial expression proteins corresponding to fnA 1 -A4, the N terminal region of fnA-D,

and fnB-D. the C terminal region of fnA-D, were gifts of Drs. Harold Erickson and Franscoise

Coussen (University of Bordeaux. Bordeaux, France). Both of these correspond to the human

protein.

Monoclonal antibody 1 /tn2 against mouse tenascin-C was a gift of Dr. Andreas Faissner

(Department ofNeurobiology. University of Heidelberg. D-69120 Fleidelberg, Germany). The

epitope for Jl/tn2 is contained on fnD of mouse tenascin-C (Gotz et al., 1996).

CSPG mixture isolated from embryonic chick brain (consisting primarily of neurocan,

phosphacan, versican, and aggrecan) was obtained from Chemicon International Inc. (Temecula,

CA). Aggrecan was from Sigma Chemical Co. (St. Louis. MO), and laminin-1 was from GIBCO

BRL (Rockville. MD). Monoclonal antibody CS-56. which reacts with the glycosaminoglycan

portion of native chondroitin sulfate proteoglycans, was from Sigma Chemical Co. Monoclonal

antibody RT97 against neurofilament was from the Developmental Studies Hybridoma Bank

(Iowa City. IA), and a polyclonal antibody against neurofilament 200 was from Sigma Chemical

Co. Monoclonal antibody tenascin-IIIB. which recognizes an epitope in fnB of human tenascin-

C. was from Chemicon.

Neuronal Cell Culture

^ 5 Cerebellar granule neuronal cultures were prepared as described by Levi et al. (1984).

Neuronal cultures were cultivated from postnatal day 8 (P8) rat pups. Brains were removed into

a Petri dish containing 5 ml of BME with 2M HEPES buffer (BME-HEPES). Cerebella were

removed, and meninges and blood vessels were peeled off and discarded to ensure minimal

contamination from endothelial cells. Cerebella were then minced into fine pieces (<0.5 mm)

with dissecting knives and incubated in BME-HEPES containing 0.025% trypsin for 10 minutes

at 37 °C. Following incubation, the trypsinization was halted by adding one ml of BME

containing 0.025% soy bean trypsin inhibitor and 0.05% DNase I. The tissue was then gently

triturated through a fire-polished pasteur pipette until it was dispersed into a homogeneous

suspension. The suspension was transferred into a fresh tube. DMEM-25 mM KCl/10% heat

inactivated FCS (3-4 ml) was added to any remaining tissue clumps, and the trituration was

repeated. Cells were then filtered through an ethanol-sterilized 40 μm mesh and centrifuged for

10 minutes at 1500 rpm. The pellet of cerebellar granule neurons was resuspended in DMEM-25

mM KCl/10% FCS and used for neurite guidance and neurite outgrowth assays as described

below.

Neurite Guidance Assay

Neurite guidance is operationally defined as directed neurite movement which is

significantly different from chance. The two most frequently used guidance assays are the stripe

assay (Vielmetter et al., 1990) and the spot assay (Snow et al., 1991 ). However, in neither has

neurite behavior been quantified. We therefore modified the spot assay to quantify the behavior

of neurites at an interface created between PLL and tenascin-C FN-III expression proteins, native

tenascin-C splice variants, or CSPGs. The PLL-protein interface was created by placing a 5 μl

drop of the protein of interest (300 nM in HanlJs buffered salt solution. HBS) in the center of a 12 mm PLL-coated glass coverslip. Coverslips in 24- well trays were incubated with the protein

drop for 2 hours at 37°C, and excess protein solution was rinsed away with HBS. Similar coating

efficiencies between the tenascin-C splice variants and expression proteins (about 5 pmole/cm²)

were verified by incubating entire coverslips with proteins conjugated to NHS-fluorescein (Pierce

Chemical, Rockford, IL). Coated proteins were removed after 2 hours by adding 2% SDS. The

fluorescence of proteins bound to PLL-coated glass was then assessed in a Cytofluor II

fluorescence microplate reader (PerSeptive Biosystems, Framingham, MA) as we have described

previously for proteins bound to cellular monolayers (Meiners et al.. 1999). In agreement with

the results of others (Dorries et al., 1996; Fischer et al., 1997), no major differences in coating

efficiencies could be observed.

Cerebellar granule neurons were plated onto the coverslips at a density of 60,000

neurons/well and cultured for 48 hours in DMEM-25 mM KCl/10% FCS. At this time,

coverslips were fixed with acetic acid/ethanol (5%/95%) for 5 minutes at -20°C. Following

fixation, coverslips were rinsed in PBS (pH 7.4. 0.14 M NaCl, 2.7 mM KC1. 1.5 mM KH₂P0₄.

4.3 mM NaHP0₄) and incubated with the appropriate primary antibody against the protein in the

drop (polyclonal full-length tenascin-C antibody for native tenascin-C splice variants and

expression proteins, or monoclonal antibody CS-56 for CSPGs) diluted 1 : 100 in PBS containing

10% FCS (PBS-serum). After rinsing in PBS. the coverslips were incubated with fluorescein-

conjugated secondary antibodies diluted 1 : 100 in PBS-serum (goat anti-rabbit secondary

antibodies for tenascin-C spots and goat anti-mouse secondary antibodies for CSPG spots)

(Organon-Technika Cappel. Durham, NC). The coverslips were again rinsed in PBS. and those

containing tenascin-C spots were incubated with monoclonal antibody RT97 against

neurofilament followed by a rhodamine-conjugated goat anti-mouse secondary antibody, whereas

those containing CSPG spots were incubated with polyclonal antibody against neurofilament 200 followed by a rhodamine-conjugated goat anti-rabbit secondary antibody. All primary and secondary antibody incubations were for 30 minutes at 4°C. Coverslips were rinsed in PBS

followed by ddH₂O and then mounted in Fluoromount-G (Southern Biotechnology Associates,

Birmingham, AL) on microscope slides. Nonspecific binding of secondary antibodies was

controlled for by omitting the appropriate primary antibody in parallel cultures.

Cultures were examined using a Zeiss Axioplan microscope equipped with an

epifluorescence illuminator with appropriate filter sets to visualize the fluorochromes. Images

of the cultures were captured using a Macintosh Quadra 700 with a Scion LG-3 frame grabber

board. Images were analyzed by counting the number of neurites on both sides of the

PLL/protein interface that either remained on their substrate (by virtue of either stopping or

turning at the interface) or crossed to the other side. A sample of 75 neurites was considered for

each side of the interface for each condition. Figure 2 is a schematic diagram presenting our

modified neurite guidance assay. Only single, nonfasiculated neurites within 10 μm of the

protein/PLL interface were considered for the analysis. This distance was chosen because

filopodia have been shown to extend 10-50 μm (Gomez and Letourneau. 1904). ]_n addition, only

neurites moving toward the interface were counted (the angle between the neurite and the

interface was less than 90°). and no neurite whose soma was sitting on the interface was counted.

The percentage of neurites that crossed from PLL to the protein of interest or from the protein

to PLL was then assessed.

Neurite Outgrowth Assay

To investigate the neurite outgrowth promoting properties of fnA-D vs. fnA-D (-) C.

PLL-coated glass coverslips in 24-well trays were incubated with expression proteins (250 nM

in HBS) for 2 hours at 37°C. In some experiments, coverslips were incubated with a mixture of fnA-D and monoclonal antibody Jl/tn2 (75 μg/ml). Excess protein solution was rinsed away

with HBS, and cerebellar granule neurons were plated onto the coverslips at a density of 60,000

neurons/well and allowed to extend neurites for 48 hours in DMEM-25 mM KCl/10% FCS. The

extent of neurite outgrowth was then determined via carboxyfluorescein diacetate (CFDA)

labeling (Petroski and Geller. 1994). CFDA (Sigma Chemical Co.) intensely stains the soma and

all processes of cultured, living neurons. Images of the cultures were captured using a Macintosh

Quadra 700 and analyzed with the NIH Image Software (available at http://rsb.info.nih.gov/).

A sample of 100 neurons with processes equal to or greater than one cell soma was considered

for each condition. The length of each primary process and its branches was measured for each

neuron, and the total neurite length was calculated as the sum of the lengths of individual

neurites.

Neurite Guidance Assay on Cellular Substrates

To investigate regulation of neurite guidance in a cellular context, we generated cellular

interfaces between untransfected BHK cells, which express no tenascin-C, and transfected BHK

cells, which over express either the largest or smallest tenascin-C splice variant (Aukhil et al..

1993), according to a modified method of Powell et al. (1977). First, transfected BHK cells were

labeled with the red fluorescent cell linker PKH26 (Sigma Chemical Co.) according to the

manufacturer^'s instructions. This dye binds irreversibly within the membranes of cells by

selective partitioning with no apparent transfer of the label to unlabeled cells (Ford et al.. 1996).

Single cell suspensions of transfected cells and untransfected cells were then mixed in a 1 : 10

ratio. The cell mixture was plated onto PLL-coated glass coverslips in 24-well trays at a density

of 1 x 10⁵ cells per coverslip. This density yielded confluent monolayers 24 hours later with

readily distinguishable "islands" of individual PKH26-labeled, transfected cells interspersed amongst the untransfected cells. The transfected cells were also readily distinguished from untransfected cells by tenascin-C immunoreactivity.

Cerebellar granule neurons were plated onto BFIK monolayers in DMEM-25 mM

KCl/10% FCS and were allowed to extend neurites for 48 hours. At this time, neurons and their

processes were labeled with CFDA. Images of the cultures were captured, and neurite behavior

was analyzed on both sides of the interface formed between an untransfected cell and a

transfected cell. The number of neurites that originated on an untransfected cell and either

remained on the untransfected cell or crossed to a transfected cell was assessed, as was the

number of neurites that originated on a transfected cell and either remained on the transfected cell

or crossed to an untransfected cell. A sample of 75 neurites was considered for each of these

conditions. Only neurites within 10 μm of the interface were included in the analysis.

Antibody Blocking Experiments on Cellular Substrates

To investigate the role of specific FN-III sequences in the regulation of neurite guidance

by cellular tenascin-C. blocking experiments were conducted using polyclonal antibodies against

full-length tenascin-C. alternatively spliced domains fnA-D. and universal domains fnl-5.

Monoclonal antibody Jl/tn2. which reacts within fnD of fnA-D. was also employed in blocking

experiments. Mixed monolayers containing untransfected and transfected BHK cells were

incubated with 75 μg/ml of antibody in DMEM-25 mM KCl/10% FCS for 1 hours at 37°C.

Cerebellar granule neurons were plated onto the cells and cultured for 48 hours in the presence

of antibodies. Neurite behavior at the interface between transfected and untransfected cells was

then evaluated. Results

The Alternatively Spliced Region of Tenascin-C Provides Permissive Neurite Guidance

Cues.

FnA-D avidly promotes neurite outgrowth from a variety of CNS neurons (Meiners and

Geller, 1997). We therefore investigated whether fnA-D can also provide guidance cues to

growing neurites. Cerebellar granule neurons were cultured for 48 hours on PLL-coated glass

coverslips containing spots of alternatively spliced or universal tenascin-C FN-III domains. The

behavior of the neurites was then analyzed at the protein/PLL interface. Figure 3 A shows that

cerebellar granule neurites demonstrated a strong preference for fnA-D when encountering an

interface between fnA-D and PLL. The neurites (which showed with a red color), and the

fnA-D/PLL interface (the fnA-D region showed as a green color) are visualized in the black and

white Figure 3 B. More than 80% of the neurites originating on PLL crossed to fnA-D, and less

than 20% of the neurites originating on fnA-D crossed to PLL. This was significantly different

from results obtained with neurites growing across a control fluorescein-labeled BSA/PLL

interface, where about 50% of the neurites originating on PLL crossed to BSA and vice versa.

The same 50/50 crossing ratio was observed for neurites growing across an imaginary interface,

created by drawing an ink circle approximating the size of a 5 μl protein drop on the back of the

PLL-coated coverslip (data not shown). The increased number of neurites crossing onto fnA-D

indicates that the alternatively spliced region provides permissive neurite guidance cues. In

contrast, universal FN-III domains fnl -5 and fn6-8 did not elicit neurite behavior which differed

significantly from the control.

Because fnA-D promotes neurite outgrowth as an expression protein and as a part of the

largest tenascin-C splice variant (Meiners and Geller. 1997). we investigated its ability to guide neurites in the context of native tenascin-C. Neurites were allowed to choose between PLL and

either the largest or smallest tenascin-C splice variant. We only assessed neurite behavior on the

PLL side of the interface as very few neurons adhered to purified tenascin-C. Neurites

consistently avoided both splice variants (Figure 3 A). This is in agreement with qualitative

results of others showing cerebellar granule neurite deflection by spots of tenascin-C

(representing a mixture of splice variants) isolated from neonatal mouse brain (Gotz et al., 1996;

Dorries et al., 1996). Therefore, the permissive guidance properties of fnA-D were masked by

other parts of the tenascin-C molecule, indicating that tenascin-C was more inhibitory on a molar

basis than fnA-D was permissive. This observation was also reflected in dose-response curves

obtained for tenascin-C and fnA-D actions : the inhibitory effect of both tenascin-C splice valiants

and the permissive effect of fnA-D were dose-dependent with a tendency toward saturation at

100 and 300 nM, respectively (data not shown). On the other hand, the smallest tenascin-C

splice variant was always more repellant than the largest tenascin-C splice variant, with only 1 -

2% of the neurites crossing from PLL to small tenascin-C as opposed to about 10% for large

tenascin-C. Blocking large tenascin-C with a polyclonal antibody against fnA-D reduced the

percentage of neurites that crossed. This suggests that fnA-D included only in large tenascin-C

may partially overcome the boundary formed by the rest of the molecule. Given that

homogenous substrates of fn6-8 (Meiners and Geller. 1997) and large and small tenascin-C splice

variants (Chiquet and Weh le-Haller. 1994; Meiners and Geller. 1997) all promote neurite

outgrowth, the results of this experiment indicate that the ability to facilitate neurite extension

does not necessarily correlate with the ability to provide permissive neurite guidance cues.

The Alternatively Spliced Region Overcomes Tenascin-C Boundaries to Neurite Advance.

We next investigated the hypothesis that a molar excess of the alternatively spliced region could overcome the inhibition of the rest of the tenascin-C molecule. To address this issue, we

incubated PLL-coated coverslips with spots of protein consisting of a mixture of fnA-D and

small tenascin-C. The concentration of small tenascin-C was held constant at 100 nM while that

of fnA-D was increased from 100 nM to 400 nM (Figure 4 A). As in Figure 3. only 1 -2 % of the

neurites crossed onto small tenascin-C. This number increased to 8-10 % for small tenascin-C

in combination with 100 nM fnA-D. precisely the same percentage of neurites that crossed to

large tenascin-C (Figure 3). The percentage of neurites that crossed onto mixtures of small

tenascin-C and fnA-D increased as the concentration of fnA-D was increased and reached the

maximum with 300 nM fnA-D. This concentration resulted in 60-70% of the neurites crossing:

larger concentrations of fnA-D did not further increase the percentage of neurites crossing.

Hence, the concentration of fnA-D that is the most efficacious at providing neurite guidance cues

by itself (Figure 3) is also best to overcome the inhibitory guidance cues of small tenascin-C.

Neurites more readily crossed onto 300 nM fnA-D (Figure 3) than onto a mixture of 300 nM

fnA-D and 1 00 nM small tenascin-C, indicating that fnA-D largely mitigates, but does not

entirely abolish, the inhibitory properties of small tenascin-C.

We also investigated the ability of the alternati\ ely spliced region to overcome the

inhibitory guidance cues of large tenascin-C. We found that a molar excess of fnA-D weakened

the boundary formed by this tenascin-C splice variant ( 100 nM); however, the maximal effect

was observed with 200 nM fnA-D (Figure 4 B) rather than 300 nM. The lower concentration of

fnA-D necessary to overcome the boundaries formed by large as opposed to small tenascin-C

probabh reflects the fact that large tenascin-C a ead} contains one fnA-D sequence whereas

small tenascin-C contains none.

The Alternatively Spliced Region Overcomes CSPG Boundaries. Our next objective was to investigate whether the permissive guidance cues of fnA-D

could also override inhibitory guidance cues provided by other types of molecules. We

investigated its effects in combination with CSPGs. because CSPGs deflect neuronal processes

in culture (Snow et al., 1990), and because tenascin-C and CSPGs are often co-regulated on

astrocytes (Meiners et al., 1995; Powell et al.. 1997; McKeon et al., 1991). We first assessed

neurite behavior at an interface formed between PLL and a mixture of CSPGs (Table 1 )

consisting largely of neurocan. phosphacan. versican, and aggrecan. Because native CSPGs with

intact glycosaminoglycan side chains revealed a smear on SDS-PAGE gels and accurate

molecular weights could not be assigned (data not show n ), we used 10 μg/ml of the CSPG

mixture in this experiment rather than a specified molar concentration. Neurites avoided the

CSPG mixture, with only 1 -2% crossing from PLL to the CSPGs. As with tenascin-C. neurons

did not adhere to the CSPG mixture, and neurite behavior on the CSPG side of the interface was

not assessed. When the CSPGs were combined with fnA-D (300 nM), about 60% of the neurites

now crossed onto the mixture of CSPGs and fnA-D. Larger concentrations of fnA-D did not

further increase the percentage of neurites crossing.

TABLE I. FNA-D OVERCOM ES CSPG BOUN DARI ES TO N EURITE ADVANCE

% of neurites crossing to CSPG mix % of neurites crossing to aggrecan

Addition Concentrati ion Concentration

300 nM 1 μM 300 nM 1 μM

None 1 ± 1 2 ± 1

FnA-D 58 ± 4 59 ± 4 64 ± 4 63 ± 5

Laminin- 1 9 ± 1 25 ± 4 1 0 ± 2 23 ± 3 fn6-8 12 ± 2 20 ± 3 14 ± 2 21 ± 4

Data represent the mean ± SEM (n = = 4) We compared the effects of fnA-D with laminin-1, a potent promoter of neurite

outgrowth. Laminin-1 was not nearly as effective in guiding neurites by itself (45% of the

neurites crossed onto 300 nM laminin-1 (M.L.T. Mercado, unpublished data) as opposed to 80%

for fnA-D) or in overcoming the CSPG barrier (only about 10% of the neurites crossed onto the

mixture of CSPGs and laminin-1). Increasing the concentration of laminin-1 to 1 μM only

increased the percentage of crossed neurites to 25-30%. The experiment was repeated using a

single CSPG, aggrecan, instead of a mixture, and similar results were obtained. FnA-D was more

effective than laminin- 1 in mitigating the inhibitory guidance cues of aggrecan. FnA-D was also

more effective than fn6-8, another neurite outgrowth promoting molecule (Meiners and Geller,

1997) that does not provide guidance information to neurites (Figure 3 A). Thus fnA-D

specifically overcomes boundaries to neurite advance that are formed by a variety of different

CSPGs.

Neurite Guidance and Outgrowth Are Mediated by Different Sequences Within FnA-D.

Facilitation of neurite outgrowth by fnA-D bound to inert substrates has been mapped

to fnD (Gotz et al., 1996). We therefore explored the question, are neurite outgrowth and neurite

guidance mediated by the same or different sequences within fnA-D? To do this, we evaluated

the ability of monoclonal antibody Jl/tn2 to alter neurite behavior in both neurite guidance and

neurite outgrowth assays. This antibody specifically blocks the neurite outgrowth promoting site

within fnD (Gotz et al., 1996; Gotz et al., 1997: Meiners et al.. 1999). Spots of fnA-D or a

mixture of fnA-D and Jl/tn2 were made in the center of PLL-coated coverslips, and neurite

behavior was quantified at the interface. Jl/tn2 did not alter the percentage of neurites crossing

from PLL onto fnA-D (Figure 5 A) or the percentage of neurites crossing from fnA-D to PLL

(data not shown). Neurite outgrowth assays were then conducted to quantify process extension on PLL or homogenous substrates of fnA-D or a mixture of fnA-D and Jl tn2 adsorbed to PLL- coated coverslips. Box-and- whisker plots of total neurite length are shown in Figure 5 B. Boxes

enclose 25th and 75th percentiles of each distribution and are bisected by the median; whiskers

indicate 5th and 95th percentiles. As expected, neurites were considerable longer on fnA-D in

comparison to PLL, and Jl/tn2 eliminated the promotion of neurite outgrowth by fnA-D. In

control experiments, monoclonal antibody tenascin III-B. which reacts within fnB (Chemicon

International Inc.), failed to alter outgrowth or guidance by fnA-D. These results indicate that

neurite guidance by fnD is regulated by a different sequence from that promoting neurite

outgrowth.

To begin to localize neurite guidance site(s) within fnA-D to a particular region of the

protein, cerebellar granule neurons were cultured for 48 hours on PLL-coated coverslips

containing spots of fnAl-A4, the N terminal portion of fnA-D; fnB-D, the C terminal portion of

fnA-D; or a mixture of fnAl -A4 and fnB-D (300 nM of each). Neurite behavior was then

evaluated at the expression protein/PLL interface (Figure 6). Only 20% of the neurites

originating on the PLL side of a PLL/fnAl-A4 interface crossed onto fnAl-A4. while neurites

originating on the fnAl-A4 side of the interface showed no bias for either PLL or fnAl-A4

(compare to neurite behavior at the BSA/PLL control interface in Figure 3). On the other hand,

neurites showed a preference for fnB-D: the percentages of neurites crossing to fnB-D from PLL

and to PLL from fnB-D were nearly identical to those observed for fnA-D (compare to Figure

3). Hence fnB-D mimicked the actions of fnA-D. An equimolar mixture of fnA 1-A4 + fnB-D

also mimicked the actions of fnA-D. suggesting that the C terminal portion of fnA-D provides

permissive neurite guidance cues and overcomes the inhibitory boundary formed by the N

terminal portion. We next explored the hypothesis that fnC provides guidance cues to growing neurites.

The rationale for this hypothesis was based on published work demonstrating that cerebellar

granule neurites avoid rodent fnA-D expression proteins which lack fnC (Gotz et al., 1996). We

compared fnA-D to an fnA-D expression protein missing fnC (fnA-D (-) C) in neurite guidance

and neurite outgrowth assays. Neurite behavior was quantified at fnA-D or fnA-D (-) C (300

nM)/PLL interfaces (Figure 7 A). As in Figure 3 A. more than 80% of the neurites crossed from

PLL onto fnA-D. This was reduced to about 25% for neurites crossing onto fnA-D (-) C. These

data are consistent with the hypothesis that fnC provides permissive guidance cues which

overcome the barrier to neurite advance formed by fnAl-A4, the N terminal portion of fnA-D

(Figure 5). As with fnAl-A4, neurites originating on fnA-D (-) C did not show a preference for

either fnA-D (-) C or PLL. When neurite outgrowth assays were performed for neurons cultured

on fnA-D or fnA-D (-) C adsorbed to PLL-coated coverslips (Figure 7 B). both proteins were

found to be equally permissive to process extension in comparison to PLL alone. These results

along with those of Figure 6 imply that neurite guidance and neurite promotion by fnA-D are

mediated through different alternatively spliced FN-III domains: fnC for neurite guidance and

fnD for neurite promotion.

FnA-D Guides Neurites in the Context of Cellular Tenascin-C.

Work with purified substrates is informative but does not always predict the in vivo

situation, where many molecules are present in a biological matrix (Meiners and Geller. 1997).

We therefore investigated the ability of fnA-D to provide permissive neurite guidance cues in the

context of tenascin-C expressed by a cell, where a neuron would normally encounter it. BHK

cells transfected with the largest or smallest splice variant of human tenascin-C (BHK-TN.L or

BHK-TN.S cells, respectively) were combined with control, untransfected BHK cells in a mixed monolayer. Cerebellar granule neurons were cultured on the mixed monolayer for 48 h, and the

behavior of neurites at the interface formed between transfected and control cells was assessed.

Figure 8 A presents an image of the neurorJBHK co-culture following double

immunocytochemistry with antibodies against full-length tenascin-C to detect transfected cells

and RT97 to detect neurons. Neurites crossed quite readily from control BHK cells to BHK-

TN.L cells, but they avoided crossing from BHK cells to BHK-TN.S cells. Similar results were

obtained using cerebral cortical neurons (data not shown). This is in contrast to results obtained

with purified substrates of tenascin-C, which always formed barriers to neurites regardless of the

splice variant present (Figure 2).

We then quantified neurite behavior at the interface formed between transfected and

control BHK cells (Figure 8 B). BHK-TN.L or BHK-TN.S cells were labeled with the

membrane marker PKH26 to ensure that we were examining a cellular rather than a matrix

boundary. Immunocytochemistry and Western blotting demonstrated that PKH26 labeling did

not interfere with the expression of tenascin-C by the transfected cells (data not shown). Neurites

demonstrated a preference for BHK-TN.L cells in comparison to control BHK cells. About 70%

of the neurites that originated on a BHK cell crossed to a BHK-TN.L cell, and only 20% of the

neurites that originated on a BHK-TN.L cell crossed to a BHK cell. This was significantly

different from neurite behavior observed at a control BHK/BHK interface created between BHK

cells and PKH26-labeled BFIK cells, where the percentage of neurites that crossed to and from

a PKH26-labeled cell was 45-50%. On the other hand, neurites demonstrated a preference for

BHK cells over BHK-TN.S cells. The percentage of neurites that crossed from a BHK cell to

a BHK-TN.S cell (20%) was significantly lower than control, while the percentage of neurites

that crossed from a BHK-TN.S cell to a BHK cell (60-65%) was significantly higher than

control. Therefore, only small tenascin-C provides inhibitory neurite guidance cues when expressed by a BHK cell. This suggests that the alternatively spliced region included in large

tenascin-C overcomes the barrier formed by the rest of the molecule by providing permissive

neurite guidance cues of its own.

To ascertain that fnA-D does indeed provide permissive guidance cues in the context of

a cellular matrix, a panel of antibodies against tenascin-C was tested for interference with neurite

behavior at cellular interfaces (Figure 9). The selection included polyclonal antibodies against

full-length tenascin-C. fnA-D. and fnl -5, and monoclonal antibody Jl/tn2. All of these

antibodies cross-react with the large tenascin-C splice variant on transfected BHK cells. As

expected, the polyclonal antibody against fnA-D and monoclonal antibody J l/tn2 do not cross

react with the small tenascin-C splice variant, the fn 1 -5 antibody does not cross-react with fnA-

D, and the fnA-D antibody docs not cross-react with fnl -5 (Meiners and Geller. 1997).

The first antibody tested was a polyclonal antibody against full-length tenascin-C. In the

presence of this antibody, the percentage of neurites that crossed from a BHK cell to a BHK-

TN.S or a BFIK-TN.L cell w as indistinguishable from the control value obtained for neurites

crossing from BHK cells to BHK cells (compare to Figure 8 B ). as was the percentage of neurites

that crossed from a BHK-TN.L or BFIK-TN.S cell lo a BHK cell (data not shown). This

confirms that large and small tenascin-C were directly responsible for the permissive and

inhibitory neurite guidance properties of BHK-TN.L and BHK-TN.S cells, as opposed to some

other factor produced by the transfected cells. The polyclonal antibody against fnl -5 did not

alter the percentage of neurites that crossed to BHK-TN.L or BHK-TN.S cells, which was to be

expected because an expression protein corresponding to this sequence did not provide neurite

guidance cues (Fisure 3). We then examined the effects of the two antibodies that react within the alternatively

spliced region, polyclonal antibody against fnA-D and monoclonal antibody Jl/tn2. Neither of

these antibodies altered the percentage of neurites that crossing from BHK cells to BHK-TN.S

cells (data not shown). However, the polyclonal antibody against fnA-D dramatically reduced

the percentage of neurites that crossed to BHK-TN.L cells from 70% to 20%. In the presence

of this antibody, BHK-TN.L cells repelled neurites to the same extent as BHK-TN.S cells.

Therefore the permissive guidance cues of large tenascin-C expressed by transfected BHK cells

could be mapped to the alternatively spliced region, suggesting that fnA-D 's ability to guide

neurites is masked in purified tenascin-C (Figure 3) but revealed in the BHK cell matrix.

Monoclonal Jl/tn2 had no effect on the percentage of neurites crossing from BHK cells to BHK-

TN.L cells. Hence neurite promoting sequences within fnA-D do not provide guidance cues to

neurites. indicating that neurite outgrowth and guidance facilitated by the alternatively spliced

region of tenascin-C are distinct events which can be independently regulated on cellular as well

as inert substrates.

Discussion

The alternatively spliced FN-III region of tenascin-C. designated fnA-D. promotes neurite

outgrowth as a substrate-bound molecule and also facilitates neurite guidance. Its permissive

actions can be seen whether fnA-D is presented to neurons as a purified expression protein or as

part of cellular tenascin-C in a biological matrix. Other molecules, such as the netrins (Kennedy

et al.. 1994; Serafmi et al.. 1994), also have strong effects on both the outgrowth and orientation

of axons. However, in the case of the netrins. both processes are mediated through the same

neuronal receptor, which probably interacts with the same functional domain of the netrin

molecule (de la Torre et al.. 1997). To our knowledge. fnA-D is the first molecule that independently facilitates neurite outgrowth and guidance through different sequences (located

within alternatively spliced FN-III domains D and C, respectively), providing strong evidence

that outgrowth and guidance are separable events.

The fact that human fnA-D provided permissive guidance cues was somewhat surprising

at first given published data with purified substrates of mouse fnA-D (Gotz et al., 1996). Mouse

fnA-D facilitates process extension to the same extent as human fnA-D due to a common neurite

outgrowth promoting site within fnD (Gotz et al.. 1 96), but has been reported to forms barriers

to neurites. This implies that some sequence unique to human but not mouse fnA-D facilitates

neurite guidance and that the common neurite outgrowth promoting site is not involved. In

agreement with this hypothesis, monoclonal antibodies against the neurite outgrowth promoting

site within fnD did not alter the ability of human fnA-D to guide neurites. We also found that

neurites demonstrated a preference for human fnB-D in guidance assays but avoided rat fnB-D

(data not shown). The rat fnB-D expression protein used in our assay and the mouse fnA-D

expression protein used in the Gotz et al. study w ere both obtained via PCR using rodent cDNA

as the template. However, it appears both of these proteins lack the alternatively spliced FN-III

domain C. in agreement with reports demonstrating that cDNA encoding the largest splice

variant of rodent tenascin-C does not contain this domain (LaFleur et al., 1994). We found that

a naturally occurring variant of human fnA-D lacking fnC also formed barriers to neurites instead

of attracting them. Taken together, these data suggest that fnC is not only responsible for the

permissive guidance cues of human fnA-D, but overcomes inhibitory guidance cues provided by

the rest of the molecule. In contradiction to the earlier reports (LaFleur et al.. 1994; Gotz et al..

1996), PCR data have now demonstrated that alternatively spliced transcripts of mouse tenascin- C can contain fnC (Dorries et al., 1996; Gotz et al., 1997). although they are presumably less

common than mouse transcripts lacking fnC. Hence, it is of great interest to determine if mouse

fnC also imparts permissive guidance cues to neurites, or if the guidance properties of the

alternatively spliced region are unique to the human protein.

In contrast to the permissive guidance cues provided by human fnA-D, purified substrates

of all splice variants of human (see Figure 3) and mouse (Gotz et al., 1996; Dorries et al., 1996)

tenascin-C are repulsive to advancing growth cones (Dorries et al., 1996). The growth cone

repelling properties have been attributed to the EGF domains (Gotz et al., 1996; Dorries et al.,

1996). Therefore, on a molar basis, the EGF domains are more inhibitory than the alternatively

spliced region is permissive. However, a 2-3 fold molar excess of fnA-D significantly overcomes

the boundary formed not only by tenascin-C. but also by a variety of CSPGs. Much larger

concentrations of laminin-1 were not nearly as effective. This is significant in that tenascin-C

and CSPGs are up regulated on glial scars after injury (McKeon et al.. 1991 ; Pindzola et al.,

1993), where they have been strongly implicated in failed axonal regeneration (Gates et al., 1996;

Davies et al.. 1997). Full recovery cannot occur following CNS injury unless axons are guided

across the inhibitory terrain of the glial scar, suggesting a potential therapeutic role for fnA-D.

We have previously suggested that functions of the EGF domains of tenascin-C are

obscured in cellular as opposed to purified tenascin-C. perhaps by cell-derived molecules binding

to them or due to conformational restraints on cellular tenascin-C (Meiners and Geller. 1997).

Specifically, the EGF domains promoted neurite outgrowth as purified expression proteins

(Dorries et al.. 1996; Gotz et al., 1996) but had no effect on outgrowth in the context of cellular

tenascin-C (Gotz et al.. 1997: Meiners and Geller. 1997). We found that antibodies directed

against tenascin-C FN-III domains 6-8 and A-D blocked all regulation of neurite outgrowth by cellular tenascin-C; the EGF domains did not contribute (Meiners and Geller, 1997). We

therefore reasoned that the boundary-forming properties of the EGF domains, in addition to the

neurite promoting properties, might be similarly attenuated in the cellular tenascin-C. If the EGF

boundary was weakened in a biological matrix, the permissive guidance cues of fnA-D might

then be revealed in the large tenascin-C splice variant. In support of this hypothesis, transfected

BHK cells which over expressed small tenascin-C formed a barrier to neurites whereas cells

which over expressed large tenascin-C were attractive to neurites. However, more neurites

crossed onto BHK-TN.S cells than onto purified small tenascin-C, and fewer neurites crossed

onto BHK-TN.L cells than onto purified fnA-D (compare Figs. 3 and 8). At the same time, in

early experiments, neurites preferred BHK cells to which fnA-D was bound over BHK-TN.L

cells. This suggests that the boundary-forming properties of tenascin-C's EGF domains were

partially but not totally eliminated in the BHK cell environment. Monoclonal antibodies against

neurite outgrowth promoting sites did not affect the percentage of neurites that crossed to either

BHK-TN.L or BHK-TN.S cells, demonstrating that neurite guidance and outgrowth facilitated

by fnA-D were separable phenomena on cellular as well as inert substrates.

While our results were obtained using BHK cells, it seems quite conceivable that

guidance of neuronal processes by tenascin-C splice variants could vary with cell type. Different

cell type-specific molecules might bind and mask different active sites for neurite guidance

within the tenascin-C molecule, as we have seen for neurite outgrowth promoting sites within

the alternatively spliced region (Meiners et al., 1999). Alternatively, cell-type specific molecules

might provide neurite guidance cues of their own which compete with or over ride those of

tenascin-C. For example, when the ratio of CSPGs to fnA-D was low, neurites were deflected,

but as the ratio of fnA-D was increased, neurites crossed. Flence the neuronal growth regulatory

properties of tenascin-C or any other matrix protein can at best be discussed in a relative sense, and coordinated expression of specific tenascin-C splice variants by particular subsets of cells

may provide appropriate micro environments for regulated changes in neuronal process

outgrowth.

In summary, we have shown that the alternatively spliced region of human tenascin-C

contains independent domains that promote either neurite outgrowth or neurite guidance.

Extension of neurites is facilitated through alternatively spliced FN-III domain D, and orientation

of growth is influenced by alternatively spliced FN-III domain C. Each of these processes can

be regulated without affecting the other, indicating that neurite outgrowth and neurite guidance

are distinct fundamental mechanisms of neuronal growth. Moreover, the ability of fnA-D to

promote guidance was stoichiometric, and fnA-D could overcome inhibitory actions of both

tenascin-C and CSPGs. Thus. fnA-D on its own might find applicability as a reagent to promote

neurite growth in otherwise inhibitory environments.

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Example 2

We have found that the region of tenascin-C containing only alternately spliced fibronectin type-

Ill domains A-D, called fnA-D, when used by itself, dramatically increases neurite outgrowth from a variety of CNS neurons in culture. In addition, the fnA-D protein also can overcome the

inhibitory actions of chondroitin sulfate proteoglycans, which are thought to inhibit neuronal

regrowth after injury to the central nervous system. We have found that the fnA-D region of

tenascin-C, when used by itself is able to promote:

( 1 ) growth of axons

(2) extension of axons across inhibitory boundaries

To further elucidate these phenomena, we used overlapping synthetic peptides to localize the

neurite outgrowth promoting site within fnD to two 15 amino acid sequences, called D4 and D5.

An antibody against D5 blocked promotion of neurite outgrowth from cerebellar granule neurons

by fnD as well as tenascin-C. indicating that this peptide sequence is functional in the context

of the native molecule. We then evaluated the overlapping region of D4 and D5 with testing of

shorter synthetic peptides restricted the neurite outgrowth promoting site to 8 amino acids,

VFDNFVLK (SEQ ID NO: 1 ), which represents AA 1646-1653 of the human tenascin-C cDNA

(Gherzi.R.. Carnemolla.B.. Siri.A.. Ponassi,M.. Balza.E. and Zardi,L, Human tenascin gene.

Structure of the 5 '-region, identification, 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 conserved in tenascin-C sequences derived

from all the species available in Genbank. To investigate the hypothesis that "FD" and "FV" are

critical for the interaction with neurons, we tested a recombinant fnD protein and a synthetic

peptide in which "FD" and "FV" were changed to "SA" and"SS", respectively. These molecules

did not facilitate process extension, suggesting that the conserved amino acids are required for

formation of the active site in fnD. We conclude that the growth promoting region of fnA-D is within fnD. and the minimal

region we have identified is seven amino acids comprising AAs 1647-1653 of the human

tenascin-C cDNA as included in Genbank.

Example 3

We also investigated whether VFDNFVLK could be used as a reagent to overcome the

neurite outgrowth inhibitory properties of chondroitin sulfate proteoglycans (CSPGs), the major

inhibitory molecules found in the central nervous system after injury. Neurons do not grow when

they come in contact with CSPGs. When mixed with CSPGs. the peptide significantly enhanced

outgrowth on proteoglycans and was more effective than the neurite outgrowth promoting

molecules laminin-1 or Ll -Fc. thus demonstrating that fnA-D and its smaller derivatives might

find utility as part of a regeneration cocktail to stimulate neuronal regrowth following CNS

trauma.

Example 4

We hypothesized that there would be a receptor for D5 that would mediate the neurite-

outgrowth promoting actions. [31 integrins have been sho n to act as receptors for tenascin-C

mediating signals from the extracellular matrix to the cytoskeleton. For this reason, we

investigated the potential role of integrins as receptors for the neurite outgrowth promoting tenascin-C peptide VFDNFVLK. We found that a commercially-available blocking antibody

against βl integrin chain eliminated the enhancement of neurite outgrowth from cerebellar

granule neurons by VFDNFVLK as well as fnA-D, supporting a role for a βl integrin as a neuronal receptor for this sequence.

We then searched for the chain partner for the βl integrin chain. Because the α7

integrin knockout mice show defects at the my otendinous junction, which is rich in tenascin-C,

we tested the hypothesis that the α7 integrin chain is the partner for βl in the promotion of

neurite outgrowth by the VFDNFVLK. An antibody against α7 integrin eliminated the

enhancement of neurite outgrowth by VFDNFVLK and fnA-D. These results cannot be

explained by the monoclonal α7 antibody recognizing the VFDNFVLK sequence because the

antibody did not cross react with the peptide on a dot blot. Thus, the α7βl integrin is a neuronal

receptor mediating neurite outgrowth promotion by VFDNFVLK and fnA-D. Our data is the

first evidence that this receptor mediates a response to a matrix molecule other than laminin-1

and the first functional data on the role of the oJβl integrin receptor in neurons. Peripheral

neurons that successfully regenerate processes following traumatic injury dramatically increase

their expression of αVβl integrin, which may then allow them to respond to permissive substrate

molecules in their environment such as tenascin-C.

Example 5

In addition to inhibition of growth, injured areas of the central nervous system form

boundaries to the entry of regrowing axons. This property has been attributed to the upregulation

of the synthesis of proteoglycans. We created an assay for boundary formation and found that

fnA-D could allow neurons to overcome proteoglycan boundaries. The fnC region is required for this action, since a tenascin fnA-D protein without fnC did not have this property. Thus, the permissive boundary crossing ability is likelyto be within fnC.

Example 6

We propose that fnA-D or subregions can be used therapeutically to stimulate regrowth

of injured axons in the human central nervous system. This protein can be delivered

therapeutically to the area of a spinal cord lesion. The smallest possible peptide can be delivered

as an infusion directly into a lesion site via a catheter. Peptides can be delivered in combinations

with trophic agents or other agents, like caspase inhibitors, that stimulate neuronal survival, and

other agents that might stimulate neuronal growth. This can be done in combination with

therapies to limit the expression of proteoblycans in the nervous system.

Alternately, one can use gene delivery technology to have cells within the region of a lesion

express fnA-D a follows: The astrocytes or other glial cells within a lesion site are caused to

express a transgene containing the fnA-D region or subregion. The transgene for fnA-D is under

the control of a cell-type specific promoter, such as the glial fibrillary acidic acid gene promoter

for astrocytes or other appropriate promoters for expression in other cell types in the wound area.

These constructs are placed into viral vectors and injected into a lesion area. The viral vectors

can adeno-associated virus or lentivirus which can express genes in glial cells or fibroblasts. The

construct would have fnA-D being under the control of the GFAP promoter to restrict expression

to astrocytes. References of interest are: Chen H, McCarty DM. Bruce AT, Suzuki,

Oligodendrocyte-specific gene expression in mouse brain: use of a myelin-forming cell type-

specific promoter in an adeno-associated virus. J Neurosci Res 1999 Feb 15;55(4):504-13; and

Mitrophanous K, Yoon S, Rohll J, Patil D, Wilkes F, Kim V, Kingsman S, Kingsman A,

Mazarakis N, Stable gene transfer to the nervous system using a non-primate lentiviral

vector.Gene Ther 1999 Nov;6(l l): 1808-18.

Example 7

We propose that the gene delivery can be similarly used to express the α7β 1 integrin receptor in

neurons to increase the neuronal response to fnA-D. This can be accomplished with replication-

deficient Sindbis virus vectors or Herpes virus vectors which are neurotropic.

References of interest include:Wang X. Zhang GR, Yang T, Zhang W, Geller Al Fifty-one

kilobase HSV-1 plasmid vector can be packaged using ahelper virus-free system and supports

expression in the rat brain. Biotechniques 2000 Jan;28(l ):102-7; Coopersmith R, Neve RL

Expression of multiple proteins within single primary cortical neurons using a replication

deficient HSV vector. Biotechniques 1999 Dec:27(6):l 1 56-60; Navarro V. Millecamps S,

Geoffrey MC, Robert J.T, Valin A, Mallet J. Gal La Salle GLEfficient gene transfer and long-term

expression in neurons using a recombinant adenovirus with a neuron-specific promoter. Gene

Ther 1999 Nov;6( 1 1): 1884-92.

Claims

We claim:

1. A peptide comprising the 8-amino acid sequence VFDNFVLK. as defined by the one-

letter amino acid code, said peptide consisting of not more than 75 amino acids.

2. A peptide of Claim 1, said peptide consisting of not more than 50 amino acids.

3. A peptide of Claim 1, said peptide consisting of not more than 20 amino acids.

4. A peptide of Claim 1, said peptide consisting of not more than 10 amino acids.

5. A peptide of Claim 1, said peptide being the 8-amino acid sequence VFDNFVLK

unlinked to any other amino acids.

6. A method of stimulating axonal and/or dendritic growth and/or guidance said method

comprising administering a peptide of Claim 1 to a neuron.

7. A method of Claim 6 wherein the neuron is in a human.

8. A method of Claim 7 wherein the peptide is delivered to the spinal cord.

9. A method of Claim 8 wherein the peptide is delivered by infusion.

10. A method of stimulating axonal and/or dendritic growth and/or guidance said

process comprising administering a vector to an injured nervous system, said vector being

nucleic acid comprising a base sequence coding for the peptide of Claim 1.

1 1. The method of Claim 10 wherein the nucleic acid molecule is in a virus at the time of

administration.

12. A method of stimulating axonal and/or dendritic growth and/or guidance, said method

comprising administering a peptide to a neuron, said peptide being at least 7 amino acids in

length, said peptide comprising all or part of a tenascin-C region, said tenascin-C region selected

from the group consisting of fnA-D. fnD, and fnC.

13. A method of Claim 12 wherein the peptide comprises the 8-amino acid peptide VFDNFVLK.

14. A method of Claim 13 wherein the peptide is free of any sequence of tenascin-C

amino acid sequence that is both outside the tenascin-C region and exceeds 100 amino acids.

15. A method of Claim 12 wherein the peptide comprises a homologous peptide sequence

identical in length to the tenascin-C region such that, if the homologous amino acid sequence and

the tenascin-C region are aligned and consecutively numbered from the same end, and like

numbered amino acids from the two sequences are compared, there is at least N percent identity

between the amino acids of the homologous sequence and the amino acids of the tenascin-C

sequence. N being 70.

16. A method of Claim 15 wherein N is 80.

17. A method of Claim 16 wherein N is 90.

18. A method of Claim 12 wherein the neuron is in a human.

19. A method of Claim 12 wherein the neuron is human and the peptide is delivered to

the spinal cord.

20. A method of Claim 12 wherein administration of the peptide is achieved by infecting

the injured nervous system with a nucleic acid vector comprising a region coding for the peptide

or a virus comprising nucleic acid comprising a region coding for the peptide.

21. A method of Claim 12 wherein the tenascin-C region is fnA-D.

22. A method of Claim 12 wherein the tenascin-C region is fnD.

23. A method of Claim 12 wherein the tenascin-C region is fnC

24. A method of Claim 12 wherein the purpose is to stimulate axonal and/or dendritic growth.

25. A method of Claim 24 wherein the purpose is to stimulate axonal and/or dendritic

growth independent of neurite guidance.

26. A method of Claim 12 wherein the purpose is to stimulate axonal and/or dendritic

guidance.

27. A method of Claim 26 wherein the purpose is to stimulate axonal and/or dendritic

guidance independent of axonal and/or dendritic growth.

28. A peptide comprising a selected peptide region selected from the group consisting of fnA-D, fnD, fnC, and homologs to fnA-D, fnD. and fnC said peptide free of any tenascin-C

region not in the selected peptide region and exceeding L amino acids in length, a homolog of a region being identical in length to that region and if aligned end-to-end with that region having at least N percent identical amino acids with that region but less than 100 percent identical amino

acids, N being 70, L being 100.

29. A peptide of Claim 28, L being 10.

30. A method of stimulating axonal and/or dendritic growth and/or guidance said method

comprising administering a peptide of Claim 28 to a neuron .

31. A method of Claim 6, 12, or 28. wherein, prior to administration of the peptide,

nucleic acid coding for the integrin receptor is added to the neuron via a vector or virus so as to

cause the number of integrin receptors in the neuron to increase.

32. A method of making a neuron more responsive to factors that promote growth or guidance, said method comprising altering the DNA content of the neurite so that it will contain

more integrin receptors.