FFRENCH-CONSTANT, Charles (Department of Pathology, University Of Cambridge Tennis Court Roa, Cambridge Cambridgeshire CB2 1QP, GB)
VOGELEZANG, Mariette, Geertruida (MIT E17-230, 77 Massachusetts Avenue Cambridge, Massachusetts, 02139, US)
CZVITKOVICH, Stefan (Cambridge Centre for Brain Repair, Robinson Way Forvie Sit, Cambridge Cambridgeshire CB2 2PY, GB)
FAWCETT, James (Cambridge Centre for Brain Repair, Robinson Way Forvie Sit, Cambridge Cambridgeshire CB2 2PY, GB)
FFRENCH-CONSTANT, Charles (Department of Pathology, University Of Cambridge Tennis Court Roa, Cambridge Cambridgeshire CB2 1QP, GB)
VOGELEZANG, Mariette, Geertruida (MIT E17-230, 77 Massachusetts Avenue Cambridge, Massachusetts, 02139, US)
CZVITKOVICH, Stefan (Cambridge Centre for Brain Repair, Robinson Way Forvie Sit, Cambridge Cambridgeshire CB2 2PY, GB)
Claims :
1. A method of promoting neuronal regeneration or repair in damaged nervous tissue comprising; increasing the amount of integrin α9 polypeptide in one or more neural cells in said tissue.
2. A method according to claim 1 wherein the one or more neural cells are neurons and the increase in the amount of integrin α9 polypeptide promotes axonal growth or re-growth in the neurons.
3. A method according to claim 1 wherein the one or more neural cells are Schwann cells and said increase in the amount of integrin α9 polypeptide promotes migration of said cells into and/or within the central nervous system (CNS) .
4. A method according to any one of claims 1 to 3 wherein the level of integrin α9 polypeptide in the cell is increased by expressing a heterologous nucleic acid encoding the integrin α.9 polypeptide in the neural cell.
5. A method according to claim 4 wherein the integrin α9 polypeptide comprises an extracellular domain (ECD) of integrin α9
6. A method according to claim 5 wherein the extracellular domain (ECD) of integrin α9 consists of a sequence having at least 60% sequence identity to the ECD domain of human integrin cc9.
7. A method according to claim 5 or claim 6 wherein the integrin α9 polypeptide further comprises a transmembrane domain.
8. A method according to claim 7 wherein the transmembrane domain consists of a sequence having at least 60% sequence identity to the transmembrane domain of human integrin α9
9. A method according to claim 7 or claim 8 wherein the integrin α.9 polypeptide further comprises a cytoplasmic domain.
10. A method according to claim 9 wherein the cytoplasmic domain consists of a sequence having at least 60% sequence identity to the cytoplasmic domain of human integrin α9
11. A method according to claim 9 wherein the cytoplasmic domain consists of a sequence having at least 60% sequence identity to the cytoplasmic domain of human integrin α4 or integrin α5.
12. A method according to any one of claims 4 to 11 wherein the nucleic acid is comprised in a vector
13. A method according to claim 12 wherein the vector is adapted for expression in a mammalian cell.
14. A method according to claim 13 wherein the vector is adapted for expression in a mammalian neuron or a mammalian Schwann cell.
15. A method according to any one of claims 12 to 14 wherein the vector is a viral vector
16. A method according to claim 15 wherein the vector is an adeno-associated virus or lentivirus vector.
17. A method according to any one of claims 4 to 16 comprising introducing said nucleic acid into the cell.
18. A method according to claim 17 wherein the nucleic acid is introduced into the cell in vitro.
19. A method according to claim 17 comprising culturing said cell in vitro under conditions for expression of the nucleic acid so as to produce the integrin α9 polypeptide
20. A method according to claim 18 or claim 19 wherein the cell is obtained from the individual.
21. A method according to claim 20 wherein said neuron is administered to the individual following introduction of said nucleic acid.
22. A method according to claim 17 wherein the nucleic acid is introduced into the cell in vivo following administration to the individual .
23. An isolated nucleic acid encoding a chimeric integrin α9 polypeptide comprising an integrin α9 extracellular domain
24. An isolated nucleic acid according to claim 23 wherein the extracellular domain (ECD) of integrin α9 consists of a sequence having at least 60% sequence identity to the ECD domain of human integrin α
25. An isolated nucleic acid according to claim 23 or claim 24 wherein the integrin α9 polypeptide further comprises a transmembrane domain.
26. An isolated nucleic acid according to claim 25 wherein the transmembrane domain consists of a sequence having at least 60% sequence identity to the transmembrane domain of human integrin α9.
27. An isolated nucleic acid according to claim 25 or claim 26 wherein the integrin α.9 polypeptide further comprises a heterologous cytoplasmic domain.
28. An isolated nucleic acid according to any one of claims 23 to 27 which is operably linked to a heterologous regulatory element .
29. A vector comprising a nucleic acid according to any one of claims 23 to 28.
30. A vector according to claim 29 adapted for expression in a mammalian cell .
31. A vector according to claim 30 adapted for expression in a mammalian neuron.
32. A vector according to claim 30 adapted for expression in a mammalian Schwann cell .
33. A vector according to any one of claims 29 to 32 wherein the vector is a viral vector
34. A vector according to claim 33 wherein the vector is an adeno-associated virus or lentivirus vector.
35. A virus particle comprising a vector according to any one of claims 29 to 34.
36. A virus particle according to claim 35 comprising a heterologous capsid.
37. A virus particle according to claim 36 wherein said capsid is comprised of rabies G envelope protein.
38. A host cell comprising a nucleic acid according to any one of claims 23 to 28, a vector according to any one of claims 29 to 34 or a virus particle according to any one of claims 35 to 37.
39. A host cell according to claim 38 which is a mammalian cell.
40. A host cell according to claim 39 which is a mammalian neuron or Schwann cell .
41. A pharmaceutical composition comprising a nucleic acid encoding an integrin α9 polypeptide .
42. A pharmaceutical composition comprising a nucleic acid according to any one of claims 23 to 28, a vector according to any one of claims 29 to 34 or a virus particle according to any one of claims 35 to 37 or a host cell according to any one of claims 38 to 40.
43. Use of a nucleic acid encoding an integrin alpha 9 polypeptide in the manufacture of a medicament for treating nerve damage .
44. Use a nucleic acid according to any one of claims 23 to 28, a vector according to any one of claims 29 to 34 or a virus particle according to any one of claims 35 to 37 or a host cell according to any one of claims 38 to 40 in the manufacture of a medicament for treating nerve damage.
45. A method of treating nerve damage comprising administering a nucleic acid encoding an integrin α9 polypeptide to an individual in need thereof .
46. A method of treating nerve damage comprising administering nucleic acid according to any one of claims 23 to 28, a vector according to any one of claims 29 to 34 or a virus particle according to any one of claims 35 to 37 or a host cell according to any one of claims 38 to 40 to an individual in need thereof.
47. A pharmaceutical composition according to claim 41 or 42, use according to claim 43 or 44 or a method according to claim 45 or claim 46 wherein the integrin α9 polypeptide comprises an extracellular domain (ECD) of integrin α9.
48. A pharmaceutical composition, use or method according to claim 47 wherein the extracellular domain (ECD) of integrin α9 consists of a sequence having at least 60% sequence identity to the ECD domain of human integrin α9.
49. A pharmaceutical composition, use or method according to claim 47 or claim 48 wherein the integrin α9 polypeptide further comprises a transmembrane domain.
50. A pharmaceutical composition, use or method according to any one of claims 47 to 49 wherein the transmembrane domain consists of a sequence having at least 60% sequence identity to the transmembrane domain of human integrin α9
51. A pharmaceutical composition, use or method according to any one of claims 47 to 50 wherein the integrin α9 polypeptide further comprises a cytoplasmic domain.
52. A pharmaceutical composition, use or method according to claim 51 wherein the cytoplasmic domain consists of a sequence having at least 60% sequence identity to the cytoplasmic domain of human integrin α9
53. A pharmaceutical composition, use or method according to claim 51 wherein the cytoplasmic domain consists of a sequence having at least 60% sequence identity to the cytoplasmic domain of human integrin α.4 or integrin α5.
54. A pharmaceutical composition, use or method according to any one of claims 47 to 53 wherein the nucleic acid is comprised in a vector.
55. A pharmaceutical composition, use or method according to any ¬ one of claims 47 to 54 wherein the nucleic acid or vector is comprised in a host cell.
56. A pharmaceutical composition, use or method according to any one of claims 47 to 55 wherein the nucleic acid or vector is comprised in a viral particle.
57. A method of screening for a compound useful in treating nerve damage comprising: contacting a cell with a test compound and; determining the level of integrin α9 in said cell . '
58. A method according to claim 57 comprising identifying a test compound useful in treating nerve damage. |
Treatment of Nerve Damage by Modulation of Cellular Alpha9
Integrin Levels
This invention relates to materials and methods for the treatment of damaged nerves, in particular to methods and materials which increase neuronal regeneration or repair at a site of nerve injury or damage, for example through promotion of axonal regeneration or migration of Schwann cells.
The regeneration of axons in the nervous system after injury is prevented by the presence and up-regulation of inhibitory extracellular matrix molecules and oligododendrocyte-related molecules in the severed region, coupled with the low intrinsic potential of adult neurons to re-'-grow processes when lesioned. A number of approaches have been undertaken to neutralise inhibitory molecules using either blocking antibodies (Schnell et al . , 1990) or enzymes for degradation (Moon et al . , 2001; Bradbury et al . , 2002) .
After an injury to the mammalian CNS, for example, a glial scar is formed which acts as a barrier that prohibits regeneration of severed axons (Fawcett and Asher, 1999) . In one approach to improve the regenerative response of damaged CNS tracts, tissue permissive to axon regeneration (e.g. peripheral nerve or Schwann cells) is grafted to bridge the glial scar region (Bunge and
Pearse, 2003; David and Aguayo, 1981) . Axons of sensory and CNS neurons have been shown to regenerate into Schwann cell grafts after injury to the spinal cord. However, at the ends of these grafts, Schwann cells and astrocytes do not mix because Schwann cells are unable to migrate in the CNS environment. A boundary forms with Schwann cells on one side, astrocytes on the other. Regenerating axons are unable to cross these boundaries from a Schwann cell to an astrocyte environment (Plant et al . , 2001). The formation of Schwann cell/CNS boundaries has been modelled in vitro. Schwann cells and astrocytes, when put together in
culture, do not intermingle and form discrete patches with boundaries seperating both cell types (Adcock et al . , 2004). In addition, Schwann cells fail to migrate on astrocytes in vitro (Wilby et al . , 1999) . Axons growing in these cultures seldom cross boundaries from Schwann cells to astrocytes, as in vivo.
After traumatic damage to the CNS and in demyelinating conditions, such as multiple sclerosis, oligodendrocyte myelin is lost from CNS axons (McQualter and Bernard, 2006) . This causes a loss of conduction in the demyelinated axons, and the demyelinated axons frequently die. The effective treatment is to remyelinate axons (Duncan, 2005) . Cells that have the capacity for re-myelination are oligodendrocytes and Schwann cells. Schwann cells are attractive for therapeutic use because they can readily be cultured in large numbers from human patients.
However, after transplantation in demyelinated CNS, Schwann cells do not migrate through CNS tissue to reach demyelinated axons, but become walled off by CNS glia. As with the use of Schwann cells for promoting axon regeneration, the failure of Schwann cells to migrate in a CNS environment prevents their use in demyelination.
Tenascin-C (TN-C) is expressed in the core of a lesion and demarcates an area where severed axons fail to regenerate after injury to the nervous system, in particular the central nervous system (Zhang et al . , 1997; Davies et al . , 1999; Tang et al . , 2003) . TN-C is also upregulated after injury to peripheral nerves. In the CNS, reactive astrocytes, invading meningeal cells and microglia produce TN-C. TN-C promotes neurite outgrowth for a number of embryonic and neonatal neurons and part of its outgrowth enhancing activity lies within the alternatively spliced fibronectin type III domain D (fnD) .
Moreover, growth of rat cerebellar granule neurons is βl integrin dependent when cultured on peptides harbouring the fnD domain
(Meiners et al . , 2001). Similarly, embryonic chick sensory- neurons extend axons on full-length TN-C in a βl integrin dependent manner. A number of integrin binding sites have been identified in TN-C including those for α2βl, α8βl (Schnapp et al . , 1995; Varnum-Finney et al . , 1995), and α9βl (Yokosaki et al . , 1998) . α8βl integrin promotes neurite outgrowth of chicken embryonic sensory neurons on the fibronectin type III repeat 6 of TN-C (Varnum-Finney et al . , 1995) . Recently, α7βl integrin was suggested to promote neurite outgrowth from cerebellar granule neurons cultured on the fnD domain (Mercado et al . , 2004) .
α9βl integrin has also been shown to promote cell migration in human neutrophils on a TN-C peptide by inhibiting migration with a blocking antibody directed against human α9βl integrin (Taooka et al . 1999) . Moreover, ectopic expression of human α9βl integrin in CHO cells significantly enhanced cell migration on a TN-C peptide harbouring the α9βl integrin binding site compared to untransfected cells (Young et al . , 2001).
In development, α9 integrin protein has been detected in pulmonary, gastrointestinal, and vascular smooth muscles (Wang et al . , 1995) . Expression in epithelial tissue was observed, for example, in airway epithelial cells (Palmer et al . , 1993) and in the skin basal layer, whereas protein in the embryonic nervous system was only detected in the epithelium covering the choroid plexus (Wang et al . , 1995) . Mice lacking α9βl integrin develop bilateral chylothorax within 6 to 12 days after birth and die of respiratory failure (Huang et al . , 2000). However, α9βl integrin has not previously been linked with neurite outgrowth or any other activity in the nervous system.
The present inventors have shown, for the first time, that integrin α9 promotes axon outgrowth and Schwann cell migration.
Modulation of integrin α9 levels may therefore be useful in the treatment of damaged nerves .
An aspect of the invention provides a method of promoting neuronal regeneration or repair in damaged nervous tissue comprising; increasing the level or amount of integrin α9 polypeptide in one or more neural cells in said tissue.
Neural cells include neurons and Schwann cells.
The level or amount of integrin α9 polypeptide may be increased in one or more neurons to promote axonal growth or re-growth. A method of promoting axonal growth or re-growth in a neuron may comprise; increasing the level or amount of integrin α9 polypeptide in the neuron.
The growth or re-growth of neuronal axons at a site of neuronal injury or damage promotes neuronal regeneration or repair in damaged nervous tissue and is therefore useful in the treatment of damaged nerves .
The level or amount of integrin α9 polypeptide may be increased in one or more Schwann cells to promote the migration of the Schwann cells from peripheral nervous system (PNS) into the central nervous system (CNS) and within the CNS to sites of neuronal injury or damage, such as spinal cord lesions. The Schwann cells provide a growth permissive environment within tissue of the CNS which promotes axonal regeneration and remyelination.
A method of promoting the migration of a Schwann cell into or within the central nervous system (CNS) may comprise;
increasing the level or amount of integrin α9 polypeptide in the Schwann cell .
The migration of Schwann cells within the CNS promotes the regeneration of axons, thereby promoting neuronal regeneration or repair in the damaged nervous tissue, for example at a site of nerve injury or damage. Repair may include remyelination of axons within the CNS, for example in the treatment of traumatic CNS damage and demyelinating conditions such as multiple sclerosis.
Schwann cells may be exogenous (i.e. transplanted or grafted) . An increase in the level of integrin α9 polypeptide in exogenous Schwann cells implanted in the CNS, for example, promotes migration of the Schwann cells out of the implantation site (i.e. the site of lesion) along lesioned tracts in the CNS, and helps regenerating axons to grow for longer distances, resulting in improved functional recovery. Exogenous Schwann cells may originate from the host into which they are subsequently transplanted or obtained from another source .
Schwann cells may be endogenous. An increase in the level of human α9βl integrin in endogenous Schwann cells increases migration of the endogenous Schwann cells initially into the CNS and subsequently to the site of nerve injury within the CNS. The presence of Schwann cells at the site of nerve injury in the CNS increases the amount of neuronal regeneration at the site.
As described above, the damaged or injured nerve may be within the CNS. The CNS includes the brain, the spinal cord, and neurons whose cell bodies lie within, or have a primary synapse in, the brain or spinal cord. Examples of such neurons include neurons of origin of the corticospinal tract, ruborospinal tract and retinal ganglion cells and the CNS branch of sensory axons. There are also neurons whose cell body lies within the CNS, but the axon is largely in the PNS, such as the neurons of the cranial
nerves (damage to which can e.g. cause Bell's palsy) and motor neurons that innervate the musculature and whose cell bodies are in the ventral horn of the spinal cord.
The nerve injury or damage may be a spinal cord injury, for example an injury caused by assault, accident, tumour, intervertebral disc or bone abnormality, or surgery, e.g. surgery for spinal problems and/or surgery to remove tumours .
The nerve injury or damage may be CNS damage other than spinal cord injury, particularly CNS damage of the following kinds: stroke; brain injury, including (without limitation) injury caused by assault, accident, tumour (e.g. a brain tumour or a non-brain tumour that affects the brain, such as a bony tumour of the skill that impinges on the brain) or surgery, e.g. surgery to remove tumours or to treat epilepsy; multiple sclerosis; and neurodegenerative diseases, such as Parkinson's and Alzheimer's.
The damaged or injured nerve may be within the peripheral nervous system (PNS) . The PNS includes nerves outside the brain and the spinal cord and includes cranial nerves (12 pair) , spinal nerves (31 pair) , nerve plexuses, and the spinal and autonomic ganglia associated with them. Also included in the PNS are all sensory nerves and the sympathetic and parasympathetic nerves .
The amount of integrin α9 polypeptide may be increased in a cell, for example a neuron or Schwann cell, by up-regulating the expression of an endogenous nucleic acid sequence encoding an integrin α9 polypeptide. For example, the integrin α9 gene within the cell may be up-regulated by the insertion of a heterologous regulatory element to drive expression of the gene. Techniques for up-regulating the expression of endogenous genes are well known in the art (see, for example, US5641670 and US5733761) .
More preferably, the level of integrin α9 polypeptide in the cell may be increased by expressing a heterologous nucleic acid encoding an integrin α9 polypeptide in the cell.
"Heterologous" and "recombinant" indicate that the nucleic acid sequence in question does not naturally occur in a particular context, for example in a cell or an ancestor thereof, and has been introduced into that context using artificial or recombinant means, i.e. by human intervention. For example, a heterologous nucleic acid may be a nucleic acid which does not occur naturally in the cell or a nucleic acid at a particular cellular or genomic location within the cell at which it does not occur naturally.
A nucleic acid which is isolated is free or substantially free of material with which it is naturally associated such as other polypeptides or nucleic acids with which it is found in its natural environment. In other word, an isolated nucleic acid exists in a physical milieu distinct from that in which it occurs in nature .
An integrin α9 polypeptide comprises an extracellular domain (ECD) of integrin α9.
A suitable α9 integrin ECD for promoting axonal growth or Schwann cell migration may be the human α9 integrin ECD (see residues 30 to 981 of Genbank accession number NM_002207.2 GI: 52485940) or a variant or fragment thereof .
Suitable variants or fragments of the integrin α.9 ECD retain the activity of the wild-type sequence to interact with integrin βl to form a α9βl heterodimer which binds tenascin-C.
The integrin α9 polypeptide will generally be membrane-bound. For example, the integrin α9 polypeptide may further comprise a transmembrane domain (TMD) .
A TMD is a hydrophobic stretch of about 15 to 30 contiguous amino acids within a polypeptide which spans the cell membrane. TMDs may be identified in a polypeptide sequence using routine sequence analysis techniques (see for example, M. Cserzo et al Prot. Eng. vol. 10, no. 6, 673-676, 1997; K. Hofmann et al (1993) Biol. Chem. Hoppe-Seyler 374, 166; Liu et al . Comput Biol Chem. 2003 Feb;27 (1) :69-76; Martelli et al . Protein Sci . 2001 Apr;10 (4) :779-87) .
The integrin α9 polypeptide may comprise the integrin α9 TMD (residues 982 to 1002 of Genbank accession number NM_002207.2 GI: 52485940or a variant thereof) or a TMD which is not naturally associated with the integrin α9 ECD heterologous transmembrane domain (i.e. a heterologous transmembrane domain). Heterologous TMDs include TMDs from integrins other than α9, and non-integrin TMDs. Many TMDs are known in the art and may be used in chimeric integrin α9 polypeptides as described herein
In some embodiments, the integrin α9 polypeptide may comprise a cytoplasmic domain. The cytoplasmic domain may be the integrin α9 cytoplasmic domain (residues 1003 to 1035 of Genbank accession number NM_002207.2 GI: 52485940 or a variant thereof) or may be a heterologous cytoplasmic domain, for example a cytoplasmic domain from an integrin other than α9, such as integrin α4 or α5, or a cytoplasmic domain from a non-integrin polypeptide.
A variant of a polypeptide sequence may have one or more of addition, insertion, deletion or substitution of one or more amino acids in the wild-type polypeptide sequence. For example, up to about 5, 10, 15 or 20 amino acids may be altered. Such
alterations may be caused by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the encoding nucleic acid.
An amino acid sequence variant of a wild-type polypeptide sequence, may comprise an amino acid sequence which shares at least 20% sequence identity with the wild-type sequence, at least 30%, at least 35%, at least 40%, at least 45%, at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95% or at least 98%. The sequence may share at least 20% similarity with the wild-type sequence, at least 30% similarity, at least 40% similarity, at least 50% similarity, at least 60% similarity, at least 70% similarity, at least 80% similarity or at least 90% similarity.
Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PJNTAS USA 85: 2444-
2448) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl . Acids Res. (1997) 25 3389-3402) may be used. Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Worcester MA USA) .
Sequence comparisons are preferably made over the full-length of the relevant sequence described herein.
Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine .for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
A fragment of a polypeptide sequence may consist of at least 10, 20, 30, 40 or 50 contiguous amino acids of the polypeptide sequence which retain the activity of the wild-type sequence.
In some embodiments, the integrin α9 polypeptide may comprise the full-length integrin α9 sequence (i.e. integrin α9 extracellular, transmembrane and cytoplasmic domains) .
The nucleic acid encoding the integrin α9 polypeptide may be operably linked to a heterologous regulatory sequence.
Suitable regulatory sequences include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on, ecdysone or tamoxifen controlled promoters .
In some preferred embodiments in which a neuron is employed, neuronal specific promoters, such as PGK (phosphoglycerate kinase-1) promoter, neuron-specific enolase (NSE) promoter, and PDGF (platelet-derived growth factor) promoter may be employed.
In some preferred embodiments in which a Schwann cell is employed, Schwann cell-specific promoters, such as PMP22 (22 kDa peripheral myelin protein) promoter or PO (myelin protein PO) promoter, may be employed.
Nucleic acid encoding an integrin α9 polypeptide may be comprised in a vector. Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in mammalian, in particular human, cells. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication in bacterial hosts such as E. coli.
Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al . , 2001, Cold Spring Harbor Laboratory Press. Preferably, the vector is a viral vector suitable for expression in mammalian cells, in particular human cells. Suitable viral vectors include adenovirus, adeno-associated virus (AAV), for example AAV serotype 2 virus, retrovirus, lentivirus, recombinant adenovirus, 'gutless' adenovirus, herpes simplex virus, and poliovirus vectors.
Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, are described in detail in Current Protocols in
Molecular Biology, Ausubel et al . eds . John Wiley & Sons, 1992.
A viral vector may be packaged into a viral particle comprising one or more capsid proteins prior to transfection of host cells.
In some embodiments, the viral vector may be packaged into a heterologous viral particle. For example, a lentiviral vector may be pseudotyped with a rabies glycoprotein, such as rabies-G envelope protein, which provides for transduction of neurons in vivo (Mazarakis et al Human Molecular Genetics 2001 10 2109-2121)
A nucleic acid or vector as described herein may be introduced into a host cell. This may occur, for example, in vitro or in vivo or ex vivo.
Suitable host cells include neural cells such as neurons or Schwann cells, preferably mammalian neurons or Schwann cells. Bacterial cells such as E. coli may also be useful as host cells for some purposes, for example in the production of nucleic acid for use as described herein.
Techniques for the introduction of nucleic acid into cells are well-established in the art and any suitable technique may be employed, in accordance with the particular circumstances. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome- mediated transfection and transduction using retrovirus or other virus, e.g. adenovirus, AAV, lentivirus or vaccinia. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage .
Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art. In some embodiments, a marker such as GFP may be used to identify cells expressing the introduced nucleic acid. Marker genes may be comprised in the same vector as the nucleic acid encoding the integrin α9 polypeptide, or on separate vectors, which may be co-transfected into a host cell.
The introduced nucleic acid may be on an extra-chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote
recombination with the genome, in accordance with standard techniques .
The introduction may be followed by expression of the nucleic acid to produce the encoded integrin α9 polypeptide .
In some embodiments, host cells (which may include cells actually transformed, although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded integrin α9 polypeptide is produced. When an inducible promoter is used, expression may require the activation of the inducible promoter .
A host cell which expresses the nucleic acid and/or comprises the expressed integrin α9 polypeptide, for example at the cell surface, may be isolated and/or purified.
In some embodiments, host cells, in particular neural cells such as neurons or Schwann cells, may be obtained from an individual, preferably an individual requiring treatment for nerve damage .
Nucleic acid encoding the integrin α9 polypeptide may be then be introduced into the host cells ex vivo using standard transfection or transduction techniques and, optionally, cultured, isolated and/or purified, prior to implantation, grafting or administration to the individual, for example at a site of nerve damage .
In some embodiments, nucleic acid may be introduced into a cell in vivo. For example, a nucleic acid or vector may be administered to the individual such that one or more cells of the individual incorporate the nucleic acid or vector. The nucleic acid or vector may administered at or adjacent to the site of nerve damage to facilitate uptake of the nucleic acid or vector by neural cells, such as neurons or Schwann cells, at or adjacent
to a damage site . Axonal regeneration is promoted in neurons at the damage site which express the nucleic acid. Migration to damage sites within the CNS is promoted in Schwann cells which express the nucleic acid. This regeneration and/or migration may be useful in the repair of nerve damage and the improvement of nerve function.
Preferably, a cell in which the level of integrin α9 is increased as described herein also expresses βl integrin. Generally, the expression of endogenous βl integrin by cells such as mammalian neurons or Schwann cells will be sufficient for axon growth as described herein. However, in some embodiments, the cell may express heterologous nucleic acid encoding βl integrin.
In some embodiments, a cell in which the level of integrin α9 is increased as described herein may also express tenascin-C. The cell may express endogenous tenascin-C or may express a heterologous nucleic acid encoding tenascin-C. In other embodiments, the level or amount of tenascin-C in the environment surrounding a damaged axon, for example tenascin-C expressed by glial or other cells, is sufficient to promote neurite outgrowth or Schwann cell migration..
Heterologous nucleic acid encoding βl integrin and/or tenascin-C may be expressed as described above for integrin α9.
Another aspect of the invention provides an isolated nucleic acid encoding a chimeric integrin α9 polypeptide comprising an integrin α9 extracellular domain.
Preferably, the isolated nucleic acid of this aspect of the invention does not encode integrin α9 (i.e. it does not comprise the α9 ECD, TMD and cytoplasmic domains) .
The α9 integrin extracellular domain may be the human α9 integrin extracellular domain or a variant thereof. The integrin extracellular domain may be linked to a transmembrane domain. Suitable ECDs and TMDs are described in more detail above.
In some embodiments, the chimeric integrin α9 polypeptide may comprise a cytoplasmic domain, which may be the α9 integrin cytoplasmic domain or a heterologous cytoplasmic domain (i.e. a cytoplasmic domain other than the integrin α9 cytoplasmic domain) , for example the cytoplasmic domain of an α integrin other than α9 integrin.
For example, if the polypeptide comprises the integrin α9 cytoplasmic domain, the transmembrane domain may be a heterologous transmembrane domain (i.e. a transmembrane domain not naturally associated with integrin α.9) . If the polypeptide comprises a heterologous cytoplasmic domain, for example the cytoplasmic domain of an a integrin other than α9 integrin, the transmembrane domain may be the α9 integrin transmembrane domain or a heterologous transmembrane domain (i.e. a transmembrane domain not naturally associated with integrin α9) .
In other embodiments, the polypeptide may lack a cytoplasmic domain. If the polypeptide lacks a cytoplasmic domain, the transmembrane domain may be the integrin α9 transmembrane domain or a heterologous transmembrane domain (i.e. a transmembrane domain not naturally associated with integrin α9) .
Preferably, a chimeric integrin α9 polypeptide as described herein retains the activity of wild-type integrin α9 i.e. it associates with integrin βl to form a heterodimer (α9βl) which binds to the octapeptide sequence AEIDGIEL of tenascin-C.
Other aspects of the invention provide chimeric integrin α9 polypeptides encoded by nucleic acid described herein, vectors comprising a nucleic acid encoding a chimeric integrin α9 polypeptide, host cells comprising such nucleic acids or vectors and/or comprising a chimeric integrin α9 polypeptide.
Nucleic acids, vectors, host cells and polypeptides are described in more detail above.
Other aspects of the invention provide a nucleic acid encoding an integrin α9 polypeptide or a vector, viral particle or cell comprising such a nucleic acid as described herein for use in the treatment of the human or animal body, for example for the treatment of nerve damage, for example by promoting axon growth or regeneration or promoting Schwann cell migration in CNS tissue as described herein, a pharmaceutical composition comprising a nucleic acid encoding an integrin α9 polypeptide or a vector or cell comprising such a nucleic acid as described herein and the use of a nucleic acid encoding an integrin α9 polypeptide or a vector, viral particle or cell comprising such a nucleic acid as described herein in the manufacture of a medicament for the treatment of nerve damage, for example by promoting axon growth or regeneration or promoting Schwann cell migration in CNS tissue .
The nucleic acid according to these aspects may encode a chimeric or non-chimeric integrin a.9 polypeptide, as described herein.
Nerve damage may include damage to the CNS, for example spinal cord or brain damage as described above or damage to peripheral nerves .
Whether it is a nucleic acid, vector, viral particle or cell according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically
effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .
Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art . Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous . A pharmaceutical composition may thus comprise a nucleic acid encoding an integrin α9 polypeptide or a vector or cell comprising such a nucleic acid as described herein and a pharmaceutically acceptable excipient.
A pharmaceutical composition may be produced by admixing or formulating the nucleic acid, vector, or cell with a pharmaceutically acceptable excipient .
Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonic!ty and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated.
Another aspect of the invention provides a method of screening for a compound useful in increasing neuronal regeneration or repair at a site of nerve injury or damage, for example by promoting axonal growth or the migration of Schwann cells in the CNS, comprising: contacting a cell with a test compound and; determining the level of integrin α9 in said cell .
An increase in the level of integrin α9 in the presence of test compound is indicative that the test compound is useful in treating nerve damage, for example by promoting axonal growth or the migration of Schwann cells
Suitable cells include neural cells, such as neurons and Schwann cells . An increase in the level of integrin α9 in a neuron in the presence of test compound is indicative that the test compound is useful in promoting axonal growth. An increase in the level of integrin α9 in a Schwann cell is indicative that the test compound is useful in promoting migration of Schwann cells.
The level of integrin α9 in the cell may be determined by any convenient technique. For example, the level of integrin α9 in may be determined at the protein level by immunological techniques, which may include, for example, determining the binding of integrin α9 specific antibodies, or at the nucleic acid level, by northern blotting or RT-PCR techniques.
The amount of test substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to ImM or more concentrations of putative inhibitor compound may be used, for example from 0.01 nM to lOOμM, e.g. 0.1 to 50 μM, such as about 10 μM.
Test compounds may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants which contain several characterised or uncharacterised components may also be used.
Combinatorial library technology (Schultz, (1996) Biotechnol . Prog 12, 729-743) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide.
A method may comprise identifying a test compound as useful in increasing neuronal regeneration or repair at a site of nerve injury or damage, for example by promoting axon growth or Schwann cell migration. Such a compound may be useful in useful in treating nerve damage in an individual.
Following identification of a compound using a method described above, the compound may be isolated and/or synthesised.
An agent identified using one or more primary screens (e.g. in a cell-free system) as having ability to increase cellular levels of integrin α9 may be assessed or investigated further using one or more secondary screens. Biological activity, for example, may be tested in vitro by determining the ability of the compound to promote neurite growth or Schwann cell migration. Test compounds found to promote neurite growth or Schwann cell migration in vitro may be tested for activity in repairing nerve damage in animal models .
Following identification of a compound as described above, a method may further comprise modifying the compound to optimise its pharmaceutical properties.
The compound may be formulated into a pharmaceutical composition as described herein for use in the treatment of neural damage.
Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents and database entries mentioned in this specification are incorporated herein by reference in their entirety.
Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.
Figure 1 shows neurite outgrowth of PC12 cells expressing human oc9 constructs which were grown for 24 hours with or without human α9βl blocking antibodies and the mean neurite length of at least 50 neurons plotted in a graph. Neurite outgrowth assays were repeated three times. The mean length of at least 50 randomly selected neurons was analysed.
Figure 2 shows neurite outgrowth of PC12 cells expressing human α9 constructs which were seeded out on laminin and exposed to AEIDGIEL and scrambled peptides. Neurite outgrowth assays were repeated three times. The mean length of at least 50 randomly selected neurons was analysed.
Figure 3 shows neurite outgrowth of PC12 cells expressing human α9 constructs which were seeded out on TN-C and exposed to AEIDGIEL and scrambled peptides. Neurite outgrowth assays were repeated three times and the mean length of at least 50 randomly selected neurons was analysed.
Figure 4 shows adhesion of human α9/α9, α9/α4, and α9/α5 integrin expressing PC12 cells to TN-C. Differentiated PC12 cells were seeded out on lOμg/ml laminin and were assayed for one hour at 37°C for cell adhesion in the presence of anti-human α9βl antibodies, AEIDGIEL or scrambled peptides. Adhesion assays were repeated twice.
Figure 5 shows adhesion of human α9/α9, α9/α4, and α9/α5 integrin expressing PC12 cells to TN-C. Differentiated PC12 cells were seeded out on lOμg/ml TN-C and were assayed for one hour at 37°C for cell adhesion in the presence of anti-human α9βl antibodies, AEIDGIEL or scrambled peptides. Adhesion assays were repeated twice .
Figure 6 shows neurite outgrowth of PC12 cells on nitrocellulose filter implants of cortical lesions. Differentiated control, α9/α9, α9/α4, and α9/α5 integrin expressing PC12 cells were seeded out on cortical filter implants, which were removed 14 days post lesion, and were cultured for 24 hours in the presence of anti-human α9βl antibodies, AEIDGIEL or scrambled peptides.
The mean neurite length of at least 50 neurons was measured and plotted in a graph.
Figure 7 shows that the α9 cytoplasmic domain is dispensable for neurite outgrowth. Differentiated PC12 cells transfected with the human integrins α4, α5, α5/α9, α9/α9, α9/oc4, α9/α5, or α9 lacking the entire cytoplasmic domain (α9) were assessed for their ability to promote neurite outgrowth on lOμg/ml laminin, TN-C, BSA, or plastic. Neurite outgrowth assays were repeated three times. The mean length of at least 50 randomly selected neurons was analysed.
Figure 8 shows that TN-C does not promote neurite outgrowth for adult sensory neurons . Adult dorsal root ganglia explants were cultured for 72 hours on lOμg/ml laminin, TN-C, or plastic and the longest neurites of at least 25 explants were measured and the mean length plotted in a graph. Neurite outgrowth assays were repeated three times. No growth of adult DRG explants on TN-C or plastic were observed.
Figure 9 shows that TN-C does not promote neurite outgrowth for adult sensory neurons. Adult dorsal root ganglia explants were cultured for 72 hours on lOμg/ml laminin, TN-C or plastic. In half the cultures, dishes were precoated with poly-D-ornithine (PORN) . The neurite length of at least 50 neurons was measured and the mean was plotted in a diagram. Neurite outgrowth assays were repeated three times .
Figure 10 shows that ectopic expression of α9/α9 and α9/α5 integrin induces neurite outgrowth in adult sensory neurons. Adult dissociated sensory neurons were transduced with adenoviruses carrying LacZ, α9/α9, α9/α4, or α9/α5 integrin transgenes and plated on lOμg/ml laminin, TN-C, or plastic, cultured for 72 hours and stained for the neuronal marker neurofilament. Neurite outgrowth assays were repeated three
times. The mean length of at least 50 randomly selected neurons was analysed.
Figure 11 shows that Ectopic expression of human α9βl integrin promotes Schwann cell migration on TN-C. Farnesylated GFP (grey) and human α9βl integrin IRES farnesylated GFP (dark grey) transfected Schwann cells were assayed for Schwann cell migration on mouse TN-C substrate after 72 hours of culture.
Table 1 shows the Genbank reference numbers for integrin βl, integrin α4, integrin α5 and tenascin-C.
Table 2 shows the domains of integrin α9 in the sequence of NM_002207.2 GI: 52485940.
Experiments
Material and Methods
Culture of PC12 cells
PC 12 cells were grown in 10% Horse Serum/5%Fetal Calf Serum/l%penicillin streptomyocin/2mM glutamine in DMEM (GIBCO) on poly-D-lysine (PDL) (0.01% in H 2 O) coated T75 flasks (NUNC). To split cells, PC12 cells were rinsed with 0.05%EDTA-PBS and trypsinised (0.1% trypsin in PBS) for 2-3 minutes at 37 0 C. Flasks were shaken and trypsination was stopped by adding 10ml of culture medium. Cells were transferred into 15ml tubes and centrifuged for 5 minutes at lOOOrpm. The pellet was re-suspended in culture medium and cells were split 1:4 or 1:5.
For differentiation, PC12 cells were seeded out on 20μg/ml Collagen (Sigma) in 10cm NUNC dishes and maintained for 2-3 days in differentiation medium (l%Insulin Threonine Selenite/ penicillin-streptomycin/glutamine/50ng/ml NGF/DMEM : GIBCO) .
Generation of retroviruses
Human versions of α4, α.5, α9, α9/4, and α.9/5 integrin were cloned into a retroviral PLIXN backbone (Stratagene) containing a Neomycin resistance gene. Packaging cells (GP+86) , which express envelope proteins that exclusively interact with rat cells, were cultured in T75 tissue culture flasks (NUNC) in 10%FCS/2mM L- glutamine/l%penicillin-streptomycin in DMEM and were split on 6 well tissue culture dishes and grown until 50-80% confluent. 94μl of DMEM without any additives were mixed with 6μl Fugeneβ for 10 minutes at room temperature (RT) . 2μg of DNA were placed in a corner of a Bijoux tube and the DMEM/Fugeneβ mix was added dropwise to the DNA, mixed by gentle shaking and incubated for 30 minutes at RT. The DNA/Fugene6 mix was added to 2ml medium and cells were incubated for 48 hours at 37 0 C. Cells were trypsinised and packaging cells of two 6 well dishes were seeded out in one T75 flask and cultured in selection medium (10%FCS/l%L-glutamine/ l%penicillin-streptomycin/DMEM plus lmg/ml G418) . Colonies of transduced cells were visible after 7days of selection and were split into new flasks and grown under selective conditions until cells are confluent. Next, 10ml medium without G418 was added to packaging cells and was collected after 48 hours, sterile filtered (pore size 0.45μm), and quick frozen on dry ice. The viral titre was determined on NIH 3T3 cells .
Retroviral transduction of PC12 cells PC12 cells were grown in 10%Horse Serum/5%Fetal Calf
Serum/l%penicillin streptomyocin/2mM glutamine/DMEM (GIBCO) . Cells were split into PDL-coated 6 well dishes to obtain 60-70% confluency. Retroviruses were defrosted at 37 0 C and polybrene (4μg/ml) was added to viruses used for transduction. 2ml retroviruses were added per 6 well dish and incubated over night (o/n) at 37 0 C. The next day, viruses were removed and fresh medium was added for further 24 hours. Cells from one 6 well were split into a PDL-coated T75 flask (NUNC) and lmg/ml G418 was applied with culture medium. After 8-10 days colonies started to appear in flasks, which were subsequently trypsinised and
replated into new PDL-coated T75 flasks (NUNC) and maintained in selection medium.
Extraction of differentiated PC12 cells The supernatant of collagen-differentiated PC12 cells was removed and cells were washed once with PBS and incubated for 30 minutes on ice with 0. lmg/ml NHS-LC-biotin (PIERCE) in ice-cold PBS. Biotinylated cells were washed three times with cell wash buffer (5OmM Tris-HCl pH 7.5, 0.15M NaCl, ImM CaCl 2 , ImM MgCl 2 ), removed from the dish with a cell scrapper and extracted with 300/xl extraction buffer (2OmM Tris-HCl pH 7.5, 15OmM NaCl, 1OmM EDTA, l%Triton X-100, complete protease inhibitor [Roche], pepstatin A) for 20 minutes on ice. Cell extracts were centrifuged with 14000 rpm for 10 minutes at 4 0 C. Supernatants were measured for protein content (Biorad) and insoluble pellets were stored at -8O 0 C. To determine protein concentration of extracted cells, a standard curve was created by measuring duplets of 'protein stained BSA standards ranging from 0 to 20μg/ml in a spectrometer at 750nm. Extracts were diluted 1:1 with H 2 O, and simultaneously treated and measured with BSA controls. Protein concentrations were determined by comparing spectrometric outputs with the BSA standard curve.
Adhesion assays 96 well plates were incubated with lOμg/ml laminin or TN-C over night (o/n) at 4 0 C. The next day, laminin and TN-C substrates were washed twice with PBS and blocked for one hour with 3% heat- denatured bovine serum albumin (BSA) (GIBCO) . Plates were washed one more time with PBS and culture medium. All concentrations and conditions were done in triplicates. 5xlO 4 cells (equals lOOμl) were seeded out and were allowed to adhere for one hour at 37 0 C. Cells were assayed in the presence of 0.3mM AEIDGIEL or scrambled TN-C peptides, and lOμg/ml mouse α-human α9βl blocking antibody (CHEMICON) . Subsequently, cells were washed two times with DMEM and fixed in ice-cold methanol for 15 minutes and subsequently
stained with 200μl of a 0.2% crystal violet solution in 2% ethanol for 5 minutes. Wells were once rinsed with destilled H 2 O and crystal violet stain was solubilized in 50μl of a 1% sodium dodecyl sulfate (SDS) (Sigma) solution. Adhesion was quantified by measuring the absorbance at 570nm. All experiments were repeated at least twice. Statistical analysis was performed using Student's t test.
Neurite outgrowth assays on substrates 8 well tissue culture plastic chamber slides (NUNC) were incubated o/n with lOμg/ml laminin (Sigma) , chicken TN-C (CHEMICON), BSA (GIBCO), or PBS. Substrates were washed twice with PBS and once with culture medium before cells were plated out. IxIO 4 differentiated PC12 cells were seeded out on substrates and grown for 24 or 48 hours in differentiation medium containing NGF (Serotec) . Blocking peptides against α9βl integrin binding sites of TN-C (AEIDGIEL) as well as scrambled control peptides were administered at a final concentration of 0.3mM, blocking antibodies against human α9βl integrin were used at lOμg/ml. Cultures were fixed with 4% para-formaldehyde (PFA) (Merck) and either subsequently analysed under a light microscope or stained for neurofilament with a rabbit polyclonal anti-neurofilament antibody (see Chapter 3.2.3) and examined by fluorescence microscopy. Neurite length was measured by using Open Lab software. The mean of 50 randomly chosen neurites was plotted in graphs. All neurite outgrowth assays were repeated at least three times. Statistical analysis was performed using Student's t test.
Neurite outgrowth of PC12 cells on cryosections Adult female Sprague Dawley rats were sacrificed and brains were dissected out and frozen first in ice-cold iso-pentane followed by liquid nitrogen. Brains were cut into 20μτn thick coronal sections and placed on PDL coated glass coverslips. 5xlO 4 differentiated PC12 cells were seeded out on each section and cultured for either 24 or 48 hours. PC12 cells were labelled by
applying cell tracker green (Molecular Probes) for 30 minutes at 37 0 C and fresh medium was added for another 30 minutes at 37 0 C. Blocking or control peptides added to cultures were used at a final concentration of 0.3mM, human α9βl integrin blocking antibody at lOμg/ml. Samples were fixed with 4%PFA for 15 minutes at RT, washed three times with PBS and mounted in fluorosave (Calbiochem) . Sections were analysed by fluorescence microscopy and the mean neurite length of PC12 cells was examined using Open Lab software. The mean of 50 randomly chosen neurites was plotted in graphs. The mean of 50 randomly chosen neurites was plotted in graphs. All neurite outgrowth assays were repeated at least three times. Statistical analysis was performed using Student's t test.
Neurite outgrowth of PC12 cells on nitrocellulose filters implanted into cortical stab injuries
Nitrocellulose filters were surgically implanted into cortical stab injuries in adult rats using standard techniques. Implants were removed 14 days post-lesion and rinsed with 0.02%EDTA/Hanks balanced salt solution (GIBCO) , shaken for 30 seconds and maintained with 1%ITS/1% penicillin streptomyocin/1% glutamine/0.01%BSA in DMEM (GIBCO). 2xlO 4 differentiated PC12 cells were plated out on filter implants and cultured for either 24 or 48 hours. Peptides and antibodies were applied as described above. Samples were fixed with 4%PFA for 20 minutes at RT, rinsed with PBS three times and permeabilized with 0.5% Triton X-100 for 5 minutes. Filters were washed three times, blocked for one hour at RT with 3%BSA/l%Goat Serum in PBS, and incubated o/n with mouse α-β-III tubulin (Sigma) and rabbit α-GFAP (DAKO) antibodies. Samples were washed three times with 0.01%Tween- 20/PBS and incubated with secondary sheep α-mouse biotin conjugated (Amersham) and goat α-rabbit CY-3 (Amersham) labelled antibodies for one and a half hours at RT in the dark. After three more 0.01%Tween-20/PBS washes, filters were incubated with streptavidin linked FITC (Serotec) for 45 minutes at RT, rinsed again, and mounted in fluorosave (Calbiochem) . Samples were
analysed by fluorescence microscopy. These experiments were performed only once per condition. The mean of 50 randomly chosen neurites was plotted in graphs . Statistical analysis was performed using Student's t test.
Western Blot Analysis of PC12 cells and PC12 conditioned medium Differentiated PC12, α4/α4, α5/α5, α9/α9, α9/oc4, α9/α5, α5/α9, and α9woCYT cells grown in PDL coated T25 flasks (NUNC) were scrapped off the dish in 300 μl extraction buffer (15OmM NaCl, 1% NP-40, 0.5% DOC, 0.1% SDS, 5OmM Tris-HCl pb.7.5, 2OmM EDTA, complete protease inhibitor [Roche] , lOμg/ml pepstatin A) after 48 hours of culture and extracted for 30 minutes on ice. Extracts were centrifuged for 20 minutes with 14000rpm at 4°C. Supernatants were measured for protein content (Biorad) and 20μg of extract were loaded per lane.
3ml conditioned media of control and transduced PC12 cells were administered with protease complete inhibitor (Roche) and centrifuged for 5 minutes at lOOOrpm. Supernatants were concentrated for high molecular weight molecules by centrifuging them in Millipore columns with a cut off at 10OkDa for at least 20 minutes, lOOOrpm at 4 0 C. Spinning was stopped when around 250μl were left in the columns to obtain 10 times more concentrated supernatant . Protein concentrations were obtained by mixing 90μl H 2 O with lOμl sample and ImI of Coomassie Blue (pre- warmed at 37C) (Biorad) , followed by 10 minutes incubation at RT, and measurement in a spectrometer at 595nm. 40μg of protein were diluted in 5x loading buffer (2%SDS; 62.5mM Tris HCl pH 6.8; 0.05mg/ml Bromophenol Blue; in H 2 O) under reducing conditions (0.2M DTT) and boiled for 5 minutes in water. Samples were loaded on large 5% agarose gels. Proteins were separated with 8mA per gel o/n in running buffer. Gels were blotted on hybond-C membranes by wet transfer (Amersham) o/n with 175mA for at least 17 hours. Membranes were rinsed with TBS-T (0.01M Tris; 0.15M NaCl; 0.05% Tween-20) and blocked in 5% milk powder/TBS-T for one
hour at RT. Rabbit polyclonal α-TN-C (KAF14) was diluted 1:500 from the provided stock solution, rat monoclonal α-fnD (Mab 578) 1:50 (all antibodies were gifts from Andreas Faissner, University of Bochum) and rabbit IgG was used at lμg/ml . Primary antibodies were incubated for 2 hours at RT. To detect Osteopontin, 10% agarose (Invitrogen) gels were run, and membranes were stained with mouse anti-Osteopontin antibodies (CHEMICON) for 2 hours at RT. All Western Blots were performed at least two times.
Integrin constructs
To generate a human α9 construct missing the cytoplasmic domain, a HindiII restriction site was introduced 10 base pairs proximal to the ATG start codon of wild-type human α9 cDNA and a stop codon two amino acids after the transmembrane domain followed by a Notl restriction site before the GFFKR motif deleted the α9 protein. The fragment was amplified by polymerase chain reaction (PCR) using the primers: 5' GCG CGC AAG CTT (HindiII) AGC GGG CGC TAT GGG GAG 3' (sense) ; 5' GCG CGA (Notl) GCG GCC GCT TAC ATC TTC CAG AGC AGC AC 3' (antisense) . After PCR, a band of the expected size of 3kb was cut out and digested with HindiII and Notl and cloned into a pcDNA3 expression vector containing a Neomycin resistance gene and a CMV promoter. For cloning an oc5/α9 construct the α5 extracellular domain was cut out of a preexisting plasmid by a Sall-Hindlll double digest obtaining a fragment of 3.0kb which was inserted into an empty Sall-Hindlll cleaved pBluescript II SK(+) multiple cloning site. The α9 cytoplasmic domain was amplified by PCR. A HindiII site was created at the very beginning of the α9 intracellular domain changing the amino acid sequence from KMGFFKR to KLGFFKR which is the sequence for the native human α5 protein. At the 3' end of the α9 tail a Sail site was attached. The fragment was amplified with the following primers: 5' GCG CGC AAG CTT (Hindlll) GGC TTC TTT CGC CGA AGG TAC 3' (sense) ; 5' GCG CGC GTC GAC (Sail) GTG GAT CAG GTC ATG TGA CTG 3' (antisense) . The PCR fragment of the
expected size (151bp) was excised, phosphorylated with PNK kinase, and inserted by blunt end ligation into the Smal site of the pBluescript II SK (+) multiple cloning site. A Hindlll-Notl digest released a 188bp fragment which was cloned into the pBluescript vector containing the extracellular and transmembrane α5 domain thus generating a chimeric α5/α9 cDNA. The α5/α9 was cut out with Sail and cloned into the Xhol site of the pcDNA3 multiple cloning site. Orientation of α5/α9 was determined by HindIII cleavage. Both constructs were sequenced. All PCRs were performed with Ultra HighFidelity Taq Polymerase (GIBCO) to minimise mis-pairing of base pairs.
Human integrin expressing cell lines
Endotoxin-free maxi preparations of pcDNA3-α5/α9 and pcDNA3- α9woCYT yielded plasmid concentrations between 2 and 5μg/μl
(Qiagen) . For generating stable cell lines, 2xlO s PC12 cells were transfected with 2μg of DNA in a total volume of 3μl by electroporation according to the manufactures protocol (AMAXA) using a cell line nucleofactor kit. 600μl of transfected cells were plated into PDL coated T25 flask (NUNC) containing 1.5ml medium and were grown for 48 hour without selection. Cells were then selected with lmg/ml G418 until colonies appeared. Cells were split, expanded, frozen, and are stored in liquid nitrogen.
Immunoprecipitations of human α-integrins
Extraction of cells and immunoprecipitations were performed as already described above. For precipitating human α9βl integrin, 5μg of antibody was used per reaction (Chemicon) . To detect human α5/α5, a rabbit polyclonal antibody was utilised that interacts with the α5 cytoplasmic domain (Chemicon) whereas α5/α9 precipitations were performed with an immunoglobulin against human extracellular α5βl (Chemicon) .
Neurite outgrowth of AMAXA transfected PC12 cells
Differentiated PC12 cells were seeded out in 8 -well chamber slides (NUNC) treated with either lOμg/ml laminin (Sigma) , chicken TN-C (Chemicon) , BSA (Sigma), or just on plastic. 2xlO 4 cells were plated per well and were grown for 48 hours at 37 0 C. The mean neurite length of PC12 cells was analysed by light microscopy. Alternatively, cultures were fixed with 4%PFA for 15 minutes at RT and stained with a primary rabbit α -neurofilament antibody for 2 hours at RT, followed by a secondary goat α-rabbit biotinylated (Amersham) immunoglobulin and by CY3 conjugated streptavidin (Amersham) . Between all antibody incubation steps, samples were washed 3x with PBS. Neurite length was measured with a fluorescence microscope using Open Lab software. All neurite outgrowth experiments with AMAXA transfected PC12 cells were repeated at least three times. The mean of 50 randomly chosen neurites was plotted in graphs. Statistical analysis was performed using Student's t test.
RNA extraction of adult rat testis
One adult rat male Sprague Dawley rat was sacrificed and testes were removed. The shaft of the homogenizer was rinsed with RNAse
ZAP (Sigma) and DEPC treated water. A piece of testis tissue was placed into a 50ml tube (NUNC) and homogenized in 20ml solution D (4 M guanidinium thiocyanate/25 mM sodium citrate pH 7.0/0.1 M mercaptoethanol/0.5% sarcosyl) . Testis tissue was homogenized on ice until no tissue was visible anymore. First, 2ml of 2 M sodium acetate pH 4 (Sigma) and then 20 ml Phenol (water saturated, pH 6.6-8, Ambion) were added and mixed by inversion for 3-5 minutes between each step. After 4ml of chloroform: isoamyl alcohol (49:1, Fluka) were added, samples were shaken vigorously for 10 seconds and were stored on ice for 15 minutes. Samples were transferred into polycarbonate centrifuge tubes (Fisher) and were centrifuged for 20 minutes at 4°C with 8000rpm in a Sorval RC5C Plus centrifuge using a SS-34 rotor. The RNA pellet was resuspended in 300μl solution D and 750μl 100%ethanol and was transferred into a 1.5ml tube and incubated o/n at 4 0 C. RNA was centrifuged at
13000rpm 4°C and washed twice with 100% ethanol and once with 75% ethanol . The pelleted RNA was air dried and resuspended in lOOμl DEPC-treated water. RNA concentration was quantified with a spectrometer. To check the quality of the isolated rat testis RNA, a sample was run on a 1.7% agarose gel and both 18S and 28SrRNA were clearly visible with contaminating DNA migrating over the 28SrRNA band.
DNAse treatment of RNA and reverse transcription-PCR (RT-PCR) 350μg of RNA (10μl of obtained RNA extract) were added to 82.7μl H 2 O, 3.3μl 3M Sodium Acetate pH 5.5 (Ambion) , lμl 0.5M MgSO 4 , lμl RNAse inhibitor and 2μl DNAseI (20 units) (Roche) and incubated for 15 minutes at 37 0 C to digest contaminating DNA and for 5 minutes at 7O 0 C to inactivate DNAseI. lμl of the mix (equals 3.5μg RNA) were run on a 1.7% agarose gel to check if DNAseI treatment was successful and has removed all DNA. For reverse transcription, lμl RNA (3.5μg) was mixed with lμl Oligo dT primers, lμl dNTPs (1OmM) and 7μl H 2 O and were incubated for 5 minutes at 65°C and 1 minute on ice. Then, 4μl 5x reverse transcription buffer, 4μl MgCl 2 (25mM) (Sigma), 2μl DTT (0.1M) and lμlRNAse inhibitor were added, mixed, and incubated for 2 minutes at 42°C. lμl (2000 units) of superscript reverse transcription enzyme was added and samples were kept for 50 minutes at 42 0 C. To inactivate the enzyme, tubes were incubated for 15 minutes at 70 0 C and to remove RNA, lμl of RNAse H was added and was allowed to digest for 20 minutes at 37 0 C. Single stranded DNA (ssDNA) was stored at -8O 0 C.
To generate probes for rat α9 integrin, primer pairs were generated and used in PCR reactions. The pair of primers I used to make the probe were: 5' CAG ACG GTA TAC TTA CCT GGG C 3' (sense) and 5' CAG CCG TCA GAT TGT AGT TCA G 3' (anti-sense) and gave a product at the expected size of 830bp. A second pair of primers: 5' CTG AAC TAC AAT CTG ACG GCT G 3' (sense) and 5' GCT GAG AAT TTC CTC TTC TCC 3' (anti-sense) was used and gave a
product at the expected size of 683bp.In detail, 1.5μl ssDNA, 5μl 1Ox buffer, 1.5μl MgCl 2 (5OmM), lμl dNTPs (1OmM), 5μl primerl (5μM) , 5μl primer2 (5μM) , 0.3μl Taq polymerase (Biotaq) and 41μl H 2 O were mixed to a total volume of 50μl and amplified in a PCR machine (Biorad) with an extension temperature of 58 0 C for 30 seconds. As control, primers against GAPDH were used. PCR products were separated on 1.7% agarose gels and the expected band at 830bp was excised and gel purified according to manufacturers protocol (Qiagen) .
Generating the in situ hybridisation probe
The oc9 PCR fragment obtained above was used as template for the next amplification step. The sense primer had a T7 promoter sequence (TAA TAC GAC TCA CTA TAGG) attached on its 5' end whereas the anti-sense primer contained a 5' Sp6 promoter sequence (ATT TAG GTG ACA CTA TAGA) . The extension temperature was set at 7O 0 C. PCR products were separated on a 1.7% agarose gel and gel purified (Qiagen) . 200ng of DNA were in vitro transcribed with 2μl DIG-labelled NTPs, 2μl 10x buffer, lμl RNAse inhibitor, 2μl Sp6 (anti-sense) or T7 (sense) DNA dependent RNA polymerase and H 2 O in a total volume of 20μl (Roche) . The transcription mix was incubated for 2 hours at 37 0 C and the enzymatic reaction was stopped by adding 2μl EDTA (0.2 M pH 8) . The probe was but on equilibrated (AMBION) columns and centrifuged for 2 minutes at 3000rpm into an Eppendorf tube. 2μl of purified probe were run on a 1.7% agarose gel to test whether in vitro transcription was successful. All samples contain a DNA band which migrates slower than the in vitro transcribed RNA.
In situ hybridization
PO and adult male Sprague Dawley rats were sacrificed and cervical dorsal root ganglia (DRGs) were removed, embedded in OCT, quick frozen on dry ice and stored at -80 0 C. DRGs were cut into 16μm thick sections on a cryo stat under RNAse free conditions. Sections were picked up on glass slides and air dried
for 30 minutes. Tissues were fixed for 10 minutes in 4%para- formaldehyde (PFA) and washed 3x 5 minutes with PBS. For permeabilisation, sections were incubated for 10 minutes with 0.5% TritonX-100 and were subsequently washed 3x for 5 minutes in PBS. To saturate positive charges, slides were exposed to
1.4%triethanolamine/0.25%acetic anhydride in stirred DEPC treated water for 10 minutes and washed 3x for 5 minutes in PBS. Sections were incubated with 500μl of pre-hybridization mix (50% formamide/2% blocking reagent [Roche] /5x SSC [Invitrogen] ) for 3 hours at RT in a humified chamber. Meanwhile, the probe for rat α.9 integrin was incubated for 5 minutes at 85°C followed by 2 minutes on ice, and was diluted 1:500 in pre-hybridisation mix. 200μl of probe mix were added per slide, and each slide was covered with a cover slip and incubated in sealed humidified chambers o/n at 68 0 C in an oven or water bath. Coverslips were removed in 5xSSC at 68 0 C in a water bath and washed for 1 hour in 0.2xSSC (3OmM NaCl, 3mM Sodium Citrate; pH7.0) at 68°C. Slides were incubated for 5 minutes in 0.2xSSX at RT and for 5 minutes in MABS (0.1M maleic acid/ 0.15M NaCl/ 0.2M NaOH) and blocked for 1 hour at RT in 500μl blocking MABS. AP conjugated anti-DIG antibodies (1:5000 in 1% blocking MAPS) was added for 1 hour at RT and slides were first washed in 2x MABS for 30 minutes and then for 5 minutes in 0.1M trizma base/0. IM NaCl/0.05M MgCl 2 pH9.5. For detection, samples were incubated with a colour reaction mix [10ml of 0. IM trizma base/ 0. IM NaCl/ 0.05M MgCl 2 pH9.5 substituted with 2.4mg levamisole, 45 μl NBT and 35 μl BCIP] . Reactions were stopped for 5 minutes in 0.01M Trizma Base/0.001M EDTA and washed for 5 minutes in PBS. To label neurons, sections were labeled o/n at 4 0 C with rabbit anti- neurofilament (3A10) antibody and washed 3x with PBS.
Subsequently, slides were incubated with secondary biotin conjugated goat anti-rabbit immunoglobulin (Amersham) for 1 hour at RT, rinsed 3 with PBS and labeled with CY3 -linked streptavidin (Amersham) . Samples were 3x rinsed with PBS and mounted in
fluorosave (Calbiochem) . Stainings were analysed with light and fluorescence microscopy .
Culture of adult rat DRG explants Adult male Sprague Dawley rats were sacrificed and dorsal root ganglia (DRGs) were removed. All roots were cut off so that only ganglia remained. DRGs were plated on lOμg/ml laminin (Sigma) , TN-C (Chemicon) , or on tissue culture plastic and grown for 72 hours at 37°C in 1%ITS+/1% penicillin-streptomycin/2mM glutamine/ 10ng/ml NGF (Serotec) / 10ng/ml NT-3 (Calbiochem) in DMEM (GIBCO) . The longest neurite of at least 25 explants was measured and the mean of the longest neurites was plotted in a graph. These experiments were repeated three times. Statistical analysis was performed using Student's t test.
Culture of dissociated adult rat DRG neurons
Adult male Sprague Dawley rats were sacrificed and dorsal root ganglia (DRGs) were removed. All roots were cut off so that only ganglia remained. DRGs were dissociated with 0.1% collagenase in 2ml DMEM for 1 hour at 37 0 C in a 5cm dish. Digested DRGs were rinsed with 13ml DMEM and triturated in ImI culture medium (lOOμg/ml transferrin [Sigma]; 0.3% BSA [Sigma]; 2% fetal calf serum [GIBCO] ; 1% penicillin-streptomycin; 60ng/ml progesterone [Sigma]; 16μg/ml putrescine [Sigma]; 0.16μg/ml sodium selenite [Sigma] ; lOμg/ml insulin [Sigma] ; 10ng/ml NGF [Serotec] , lOng/ml NT-3 [Calbiochem] in Ham's F12 medium [GIBCO]) . After another ml of medium was added to obtain a total volume of 2ml, ImI of cells was topped on 2ml of 15% BSA (Sigma) . Samples were centrifuged for 5 minutes at 650rpm and DRG neurons were enriched in the bottom of the tube. The cell pellet was resuspended in lOOμl triturating solution (1%BSA [GIBCO]; 0.002% DNAseI [Roche]; 0.05% trypsin inhibitor [Roche] ) and diluted with culturing medium.
8 well tissue plastic chamber slides (NUNC) were o/n incubate with lOμg/ml laminin (Sigma) , chicken TN-C (Chemicon) , and BSA
(Sigma) . Substrates were laid down on untreated or poly-D- ornithine (1.5μg/ml) (PORN) (Sigma) coated wells. 2xlO 4 cells were plated out per well and cultured for 72 hours at 37°C. Cells were fixed for 15 minutes at RT with 4%PFA and were stained for neurofilament. Neurite lengths were measured under a fluorescence microscope using Open Lab software. The mean neurite length was plotted in a graph. All experiments with dissociated adult sensory neurons were done more than three times. Statistical analysis was performed using Student's t test.
Adenoviral transduction of adult DRG neurons
Dissociated DRG neurons of one adult rat (around 5xlO 4 cells) were counted and seeded out on 4 poly-D-lysine coated wells (NUNC) obtaining 12.000-15.000 cells per well and were cultured o/n. Next day, 100 plaque forming units/cell of lacZ, α9/α9, α9/α4, or α9/α5 containing adenovirus were added to neurons and incubated for 48 hours at 37°C. Cells were re-plated on lOμg/ml laminin (Sigma), chicken TN-C (Chemicon) , and tissue culture plastic. Neurons were chilled down for 15 minutes on ice, washed for 5 minutes with Hanks balanced salt solution (without Mg 2+ and Ca 2+ ) (GIBCO) , gently resuspended in culture medium, and seeded out on substrates. Neurons were cultured for 72 hours and fixed in 4%PFA for 15 minutes at RT. Cells were washed 3x with PBS and blocked with 3%BSA/0.1% Tween-20 in PBS for 1 hour at RT. The primary rabbit polyclonal α-neurofilament antibody was added for 2 hours at RT and cells were washed 3x with 0. l%Tween-20/PBS . The secondary goat α-rabbit biotinylated antibody (Amersham) was incubated for 1 hour at RT and cells were labelled with CY3 conjugated streptavidin (Amersham) for 45 minutes at RT after 3 more washes. Neurons were rinsed again 3x in 0. l%Tween-20/PBS and mounted in fluorosave (Calbiochem) . Samples were analysed by fluorescence microscopy and neurite length was measured using Open Lab software . All these experiments were performed for at least three times.
Live-labelling of adenoviral transduced adult DRG neurons with an α9βl antibody
Dissociated adult DRG neurons were transduced with either lacZ, α9/α9, α9/α4, or α9/α5 integrin containing adenovirus and grown on lOμg/ml Laminin, TN-C, or on plastic as described above. After 72 hours of culture, cells were washed with 2%FCS in LIF medium (GIBCO) and the primary mouse α-human α9βl (5μg/τnl) (CHEMICON) antibody (in 2%FCS/LIF) was added to neurons for 20 minutes at RT. Cells were rinsed 3x with 2%FCS/LIF and stained with a secondary sheep α-mouse biotinylated immunoglobulin (Amersham) for 20 minutes at RT. Cells were rinsed and incubated with CY3 conjugated streptavidin (Amersham) for 20 minutes at RT and after 3 more washes with 2%FCS/LIF were fixed for 2 minutes at -20 0 C with ice-cold methanol. Fixed neurons were washed 3x with 0.1%BSA/PBS and stained with a rabbit α-neurofilament antibody o/n at 4 0 C and incubated with a goat α-rabbit FITC labelled antibody for 1 hour at RT. Between all antibody steps, cells were rinsed with 0. l%Tween-20/PBS. Neurons were mounted in fluorosave (Calbiochem) and were analysed by fluorescence microscopy. Life- labelling of transduced neurons was repeated twice.
Immunoprecipitation of transduced adult DRG neurons for ectopic human α9 proteins
Adult DRGs from adult Sprague Dawley rats were dissociated and enriched for neurons as described above. All neurons obtained from one adult rat were seeded out on 5cm NUNC tissue culture dishes coated with 1.5μg/ml poly-D-ornithine (Sigma) and lOμg/ml laminin (Sigma) . Cells were grown in culture medium (see above) for 7 days. Cell surface labelling and immunoprecipitation of ectopic integrin α-subunits was as described above.
Schwann cell culture
Immortalised Schwann cell have been cultured in 10%FCS (GIBCO) containing Dulbecco's Modified Eagle Medium (GIBCO) supplemented
with ^penicillin-streptomycin, 0.1% bovine pituitary extract (BPE) and 0.1% Forskolin (GIBCO) . Cells were passaged once a week and grown on poly-D-lysine coated tissue culture dishes (NlMC) at 37 0 C in a humidified incubator.
Transfection of Schwann cells
Cells were transfected with lentiviral constructs containing fGFP or α9βl integrin IRES fGFP under the control of the human CMV promoter using an AMAXA. electroporator and AMAXA nucleofactor kit for cell lines according to manufacturer's protocol (AMAXA) .
Migration Assay- After transfection, cells were cultured for 48 hours on poly-D- lysine (PDL) coated tissue culture flasks and split onto PDL- coated broken coverslips. Schwann cells were grown on cover slips until confluency and were inverted onto 3μg/ml mouse TN-C. Schwann cells were allowed to migrated for 3 days at 37 0 C in a humidified incubator. Next, cells were fixed by addition of 4% paraformaldehyde (PFA) to living cultures (final concentration: 2% PFA) , rinsed three times with phosphate buffered saline (PBS) pH 7.4 and blocked for 1 hour in 3%BSA (Sigma) /10% goat serum (GIBCO) /0.2% Triton X-100/PBS. Cells were incubated with a primary rabbit anti-GFP antibody (Molecular Probes) overnight at 4 0 C and secondary goat anti-rabbit biotinylated antibody (Amersham) for 1 hour at room temperature. Staining was visualised using a DAB kit (Vectorshield) according to manufacturer's protocol. Positive cells were analysed for migration from the edge of the glass cover slip using a grid with lOOμm segments.
Results
Verification of integrin expression
To assess the question whether the α4 cytoplasmic domain can induce neurite outgrowth on the α9βl ligand TN-C when fused to the extracellular α9 domain, retroviral constructs were generated
that contained full-length human α9 (α9/α9) cDNA or chimeric constructs consisting of the human extracellular α.9 domain and the cytoplasmic α.4 (α9/α4) or α5 (α9/α5) tail. PC12 cells were transduced with retroviruses harbouring those integrins and were analysed for expression of endogenous and ectopic α9 integrins.
Primers against rat α9 were generated and RNA extracts of rat testis and PC12 cell were analysed by reverse transcription-PCR (RT-PCR) . In rats, testes are the only tissue besides the injured nasal mucosa that has been reported to express α9 integrin and were therefore used as positive control. Two different primer pairs were used which detected α9 RNA at the expected size of 683 and 830 base pairs (bp) in rat testis extracts, whereas no bands were obtained from PC12 cell RNA indicating that PC12 cells do not produce α9. GAPDH PCRs yielded a band at 380bp in testis and PC12 cell preparations and confirmed that the RNA was not degraded.
Cells were biotinylated and immunoprecipitated against human α9βl integrin to examine cell surface bound integrin receptors.
Importantly, the antibody used to detect human α9βl interacts with extracellular domain of the protein. Control PC12 cells did not label positive for α9 whereas α9/α9, α9/α4, and α9/α5 transduced cells clearly expressed the human α-integrin. In general, levels of cell surface bound α9 protein were similar although α9/α9 expressing cells contained marginally higher levels than α9/α4 and α9/α5 transduced cells.
Neurite outgrowth of retrovirally transduced PC12 cells on chicken TN-C
Next, PC12 cells transduced with human α9/α9, α9/α4, and α9/α5 integrin were analysed for neurite outgrowth on lOμg/ml laminin, TN-C, BSA, or on plastic after 24 hours of culture.
All the PC12 cells grew equally well on laminin implying that the neurons were healthy and viable. PC12 cells failed to adhere to TN-C, BSA, or plastic and those few cells that attached did not form neurites or only very short stumps (TN-C: 6.6± 1.0; BSA: 6.4+ 1.4; plastic: 8.1+ 1.7μm) (Figure 1). Notably though, more cells attached to BSA or plastic than TN-C implying that TN-C harbours anti-adhesive properties for PC12 cells. In contrast, all PC12 cells transduced with human α9 constructs (α9/α9, α9/α4, and oc9/α5) adhered well to all the substrates and extended neurites on all surfaces including TN-C (25.8+8.9; 26.6±8.1;
30.8+9.4μm), BSA (26.1±6.9; 28.4+10.3; 29.4+12.7μm) , ana plastic (28. O± 9.2; 27.9+ 9.8; 29. O± 8.4μm) independent of their cytoplasmic domain. Interestingly, administration of a human α9βl blocking antibody that binds to the extracellular domain reversed this effect and transduced cells were undistinguishable from control PC12 cells in cell attachment and neurite outgrowth (11.1+3.6; 9.5+1.6; 9.9±2.3μm) implying that these cells interact with an α9βl ligand. In summary, these results indicate that PC12 cells expressing human α9/α9, α9/α4, or α9/α5 integrin demonstrate extensive neurite growth on any substrate. Similar results were obtained when the assay time was prolonged for another 24 hours. However, no differences in growth were observed between cells expressing the α9, α4 and α5 cytoplasmic domains.
Js the TN-C a9βl binding site involved in neurite outgrowth? The AEIDGIEL blocking peptide was used in neurite outgrowth assays with control PC12, α9/α9, α9/oc4, and α9/α5 positive cells grown on lOμg/ml laminin and TN-C for 24 hours. As internal control, a scrambled peptide was applied under the same conditions.
On laminin, growth of all PC12 cells, transduced or not, was identical in the presence or absence of the AEIDGIEL (0.3mM) or the scrambled peptide (0.3M) implying that the blocking peptide
acts in a specific manner as laminin does not harbour a α9βl binding site (Figure 2) . Identical behaviour of cells plated on chicken TN-C was observed, providing indication that the blocking peptide had no effect on neurite growth and thus that the chicken TN-C-α9βl integrin interaction site does not promote neurite extension for these cells or that the peptide was degraded, or ineffective at this concentration (Figure 3) . Identical results were achieved when neurites were measured after 48 hours of culture.
Increased cell adhesion of α9/cc9, α9/α4, and α9/α5 expressing PC12 cell to TN-C
Control, α9/α9, α9/α4, and α9/α5 producing PC12 cells were tested for their ability to adhere to laminin and chicken TN-C in colorimetric short-term adhesion assays.
Control and transduced PC12 cells all attached to a similar extent to laminin which was independent of the AEIDGIEL (0.3mM) blocking peptide and the α9βl blocking antibody (Figure 4) . On TN-C, untransfected PC12 cells adhere significantly less to TN-C than α9/α9, α9/α4, and α9/α5 transduced cells. The effect of α9 integrin mediated adhesion could be reversed by applying the α9βl neutralising antibody but only partially with the AEIDGIEL (0.3M) peptide (Figure 5) indicating that the α9 extracellular domain is required for cell adhesion to TN-C.
Neurite growth of PC12 cells on coronal cryosections of adult brains
Coronal cryosections of adult rat brains were seeded with control and human α9 producing PC12 cells. Neurite outgrowth of neurons was measured in the cortical part of the brain section after 48 hours of culture . Cells were grown in the presence of AEIDGIEL or scrambled peptides, or with the human α9βl blocking antibody.
Untransfected PC 12 cells grew poorly on cryosections and extended only very short neurites (13.4± 5.9μm) . Growth of these control cells was not affected by TN-C blocking peptides (15.8±4. Oμm; 14.7+6.3) or by human α9βl neutralising antibodies (14.7+ 3.6). Interestingly, α9/α9 (44.4± 19.9μm) , α9/α4 (51.4± 16.8μm), and α9/α5 (73.3+16.6μm) expressing cells displayed significantly longer neurites on cryosections when compared with control cells. Growth was not inhibited by the AEIDGIEL or scrambled peptide but was abolished when α9βl integrin was blocked with an inhibitory antibody (17.7± 6.4μm; 18.8± 6.7μm; 18.2±6.0μm). Thus, PC12 cells expressing α9 constructs grow longer neurites on cryosections of adult brains than control cells. Interestingly, oc9/α5 producing PC12 cells display the strongest effect of all α9 constructs implying that the α5 cytoplasmic domain promotes more growth than the α4 or α9 cytoplasmic tail .
Neurite growth of PC12 cells on filter implants of cortical stab lesions Nitrocellulose filter implants were inserted into cortical stab injuries of adult female rats and removed after 14days post lesion. Control, α9/α9, α9/α4, and α9/α5 expressing PC12 cells were seeded out on the filter implants and the mean neurite length of at least 50 neurons was measured after 24 hours of culture. Only neurites of PC12 cells growing in a GFAP positive area were measured.
Control PC12 cells extended short neurites (14.9+5.3μm) on GFAP labelled astrocytes whereas α9/α9 (Fig.22d), α9/α4, and α9/α5 positive cells grew significantly longer neurites (24.5+5.8;
25.0±5.3; 28.0±6.9μm). Administration of the AEIDGIEL peptide, blocking the α9βl binding site of TN-C in the third fibronectin type III repeat, or a control scrambled peptide did not affect neurite growth of control or transduced PC12 cells on
nitrocellulose implants. This experiment shows that similarly to assays on cryosections, neurite outgrowth of cells producing different α9 constructs is significantly enhanced on adult cortical filter implants (Figure 5) and can be reduced to levels of control cells when an α9βl blocking antibody is applied, showing that neurite elongation is dependent on the α.9 integrin proteins .
PC12 cells express TN-C α9/α9, α9/α4, and α9/α5 expressing PC12 cells extended neurites on TN-C but also BSA and plastic in a α9 dependent manner providing indication that PC12 cells produce their own α9βl ligand and perpetuate their own neurite growth independent of any given substrate. Cell extracts and conditioned media of control, α9/α9, α9/α4, α9/α5, α5/α9 α9woCYT (α9 integrin lacking the cytoplasmic tail) PC12 cells, which were cultured for 48 hours on PDL, were examined for Osteopontin and TN-C expression.
Antibodies against Osteopontin did not detect any full-length or cleaved versions of the protein in cell extracts or conditioned media of control or transduced PC12 cells excluding Osteopontin as a possible candidate. Surprisingly, full length TN-C is observed in cell extracts and conditioned media of control, α9/α9, α9/α4, α9/α5, α5/α9, and oo9woCYT PC12 cells. TN-C levels were identical among cell extracts but differed in some cases in conditioned media. In particular, α9/α5 positive cells deposited significantly more TN-C into the medium compared to other cells. Blots labelled with rabbit IgG did not stain at all, proving the specificity of the TN-C antibody. Interestingly, TN-C expressed by these cells did not contain the outgrowth promoting fnD domain. These results indicate that PC12 cells synthesise the α9βl ligand TN-C and secrete it into the surrounding extracellular space thus providing indication that growth of α9 transduced PC12 cells is mediated by autocrine TN-C production.
The necessity of the aθ cytoplasmic domain for neurite outgrowth The α4 cytoplasmic domain is essential for α4βl mediated neurite growth of PC12 cells on the fibronectin fragment V120 (Vogelezang et al . , 2001) . To address the role of the human α9 tail in axon growth, two constructs were generated, one comprising the extracellular human α5 domain fused with the cytoplasmic α9 tail, and the other consisting of the extracellular and transmembrane domain of the human α9 but lacked the entire intracellular domain. In the latter construct, α9 was deleted immediately before the GFFKR motif, a truncation that has been shown to abolish α9 function in cell adhesion and migration assays on the third fibronectin type III repeat of TN-C (Young et al . , 2001). PC12 cells were transfected with human α4/α4, α5/α5, α9/α9, α9/α4, α9/α5, α5/α9, or α9woCYT (lacking the cytoplasmic domain) and stable cell lines established under constant G418 selection.
Differentiated cells were biotinylated to label cell surface proteins and immunoprecipitations for human α-integrins were performed. In blots, cell surface bound integrins were visualised. Unfortunately, cells transfected with human oc4/α4 did not express the human integrin. Extracts of α5/α5 producing cells labelled strongly for human α5 integrin whereas staining of the α5/α9 band was very weak. Importantly, α5/α5 cells were precipitated with an immunoglobulin detecting the human α5 cytoplasmic domain whereas the α5/α9 pull down was performed with an antibody against α5βl and thus can not be compared due to different affinities of the corresponding antibodies. Nevertheless, α5/α9 protein seemed to be less abundant than α5/α5. All oc9 expressing cells were analysed with a human α9βl immunoglobulin interacting with the ligand binding domain (Yokosaki et al . , 1998). α9/α9 integrin was synthesised at higher levels than α9/α4 or α9/α5 integrin. Importantly, the α9woCYT
construct was detected strongly, implying that α9woCYT forms a functional heterodimer with the βl subunit . Moreover, the α9woCYT band migrated marginally faster than α9/α9, α9/α4, and α9/α5 confirming that the cytoplasmic domain was missing. Together, all transfected PC12 cells with the exception of α4/α4 produced the respective human α-integrin and were further analysed for neurite growth .
Untransfected PC12 cells grew successfully on laminin but fail to extend a significant number of neurites on TN-C, BSA, or plastic (Figure 7) . Similarly, growth of α4/α4 and α5/α5 transfected cells on TN-C, BSA, and plastic was not observed. In agreement with results obtained from retroviral transduction experiments, α9/α9, α9/α4, and α9/α5 positive cells elongated neurites on all substrates. Interestingly, α5/α9 producing cells behaved identically to control PC12 cells and grew only on laminin but not on TN-C, BSA, or plastic. Surprisingly, cells transfected with the α9 construct lacking the entire cytoplasmic domain including the GFFKR motif retained their ability to grow neurites on all substrates including TN-C, BSA, and plastic. Thus, these results show that the human α9 cytoplasmic domain is not essential for α9βl mediated neurite growth of PC12 cells.
Neurite outgrowth of adult DRG neurons on TN-C Adult rat DRG explants were isolated and cultured for 72 hours on lOμg/ml laminin, mouse TN-C, or just on plastic. The longest axon of each explant was measured and the mean length of the longest neurites was plotted in a graph (Figure 8) .
All adult DRG explants extended processes on laminin
(699.2+183.6μm) whereas not a single explant was observed to grow neurites on mouse TN-C (Figure 8) . Similarly, only one DRG ganglion displayed neurites on plastic.
Dissociated adult rat DRG neurons were evaluated for growth on TN-C. Adult dorsal root ganglia were digested and purified for sensory neurons. Isolated cells were grown for 72 hours in dishes coated with lOμg/ml laminin, mouse TN-C, or on plastic. Substrates were laid down on either untreated or poly-D-ornithine (PORN) incubated wells. Neurons extend very short processes on poly-D-ornithine treated or untreated tissue culture plastic (66.5+24.0 and 44.8±11.7μm respectively) after 72 hours (Figure 9) . Interestingly, neuronal cells cultured in dishes coated with TN-C or TN-C plus poly-D-ornithine (42.3+13.6; 52.4±18.1μm) did not display longer neurites compared to cultures on plastic or poly-D-ornithine alone. Only laminin and laminin/poly-D-ornithine substrates significantly promoted neurite elongation (170.2±46.1; 168.3±73.3μm) . Thus, adult rat DRG explants or dissociated sensory neurons did not grow neurites on mouse derived TN-C.
PO but not adult rat DRGs express α9 integrin by in situ hybridisation α9 synthesis in DRGs was analysed by in situ hybridisation. Rat α9 sense and anti-sense probes were generated from rat testis RNA by reverse transcription-PCR (RT-PCR) . Cryosections of PO and adult rat DRGs were labelled with the α9 anti-sense and sense probe and co-stained with an antibody for the neuronal marker neurofilament .
The sense probe showed marginal unspecific staining in PO DRGs and no labelling at all in adult ganglia. In contrast, PO DRGs clearly stained positive with the α9 anti-sense probe whereas no specific signal was observed in adult DRGs. Interestingly, in neonate ganglia, all neurofilament positive neurons expressed α9, whereas some cells with neuronal appearance were negative for neurofilament but still labelled for α9. Taken together, α9 is synthesised in PO rat DRG sensory neurons and is developmentally down-regulated in adult ganglia.
Expression of a9 proteins in adult DRG neurons after adenoviral transduction
Enriched adult sensory neurons were transduced with adenoviruses containing LacZ, human α9/α9, α9/α4, or α9/α5 integrin transgenes . First, cells were examined for expression of ectopic human integrins . Transduced neurons were plated on lOμg/ml laminin plus poly-D-ornithine and cultured for 6-7 days until the dish was covered by a network of neurites . Cells were biotinylated to label cell surface bound receptors and immunoprecipitated for human α9βl.
Neurons infected with an LacZ-adenovirus did not label for human α.9 integrin but stained positive when incubated with X-GaI' substrate showing that viral infection was successful. α9/α9 transduced neurons expressed human α9 integrin whereas α9/α4 and α9/α5 infected neurons remained negative for human α9 protein implying that adenoviruses containing human α9/α4 and α9/α5 integrin did not infect their target cells or that the antibody did not recognise the altered integrin or that protein levels were too low for detection. Interestingly, human α9/α9 protein delivered by adenoviral gene transfer migrated faster in polyacrylamide gels than human α9/α9 integrin derived from retrovirally transduced PC12 cells providing indication that the human α9 integrin receptor is differently processed in adult DRG neurons than in PC12 cells.
Ectopic expression of human α9 integrin constructs induce neurite outgrowth of adult DRG neurons on TN-C LacZ, α9/α9, α9/α4, or α9/α5 expressing adult sensory neurons were assayed for neurite growth on lOμg/ml laminin, TN-C, or on tissue culture plastic. Cells were cultured for 72 hours and, after fixation, were stained for neurofilament to visualise
neurons. Neurites of at least 50 cells were measured and the mean neurite length was plotted in a graph (Figure 10) .
LacZ infected neurons successfully grew on laminin but adhered poorly to TN-C and tissue culture plastic and failed to extend processes on both substrates, indicating that adenoviral transduction per se does not cause neurite growth of adult sensory neurites on TN-C or plastic . All neurons infected with adenoviruses containing human oc9 or chimeric α9 transgenes displayed strong neurite growth on laminin, showing that neurons remained healthy over the course of viral treatment . Interestingly, α9/α9 and α9/α5 transduced neurons extended processes on TN-C and on tissue culture plastic whereas α9/α4 infected cells did not grow any neurites on both substrates and thus were phenotypically identical to LacZ expressing neurons.
Indeed, neurons exposed to α9/α4 integrin containing adenoviruses fail to express functional human α9/α4 protein by immunoprecipitation and therefore did not show any growth response on TN-C or plastic. Importantly, not all neurons that adhered to TN-C or plastic formed neurites. However, neurite growth of transduced neurons on TN-C and plastic is significantly less than on laminin (***p<0.005) . In summary, human α9/α9 and α9/α5 integrin enhances neurite outgrowth of adult sensory neurons on TN-C and plastic.
Live-labelling of transduced adult sensory neurons with the mouse α-human α9βl antibody
To establish that only successfully transduced neurons extend neurites on either TN-C or tissue culture plastic, adult DRG neurons were infected with adenoviruses harbouring LacZ, α9/α9, α9/α4, or α9/α5 integrin and were cultured on lOμg/ml laminin, TN-C, or tissue culture plastic for 72 hours. Neurons were live- labelled with the mouse α-human α9βl antibody, which only detects
the human native α9βl integrin but not fixed α9βl protein, and were co-stained for the neuronal marker neurofilament.
All neurons, whether transduced or not, adhered equally well to laminin. However, significantly fewer cells expressing human α9/α9 or α9/α5 integrin attached to TN-C or plastic than to laminin providing indication that not all cells were successfully transduced and that only a subset of neurons expressed α9/α9 or α9/α5 integrin. However, when oc9 integrin infected cells were plated on laminin virtually all of them stained positive for human α9 integrin, indicating that only neurons expressing high levels of human α9/α9 and α9/α5 integrin attached to TN-C or plastic .
Neurons transduced with adenoviruses containing human α9/α4 integrin failed to attach to TN-C and plastic and did not stain positive for human α9βl integrin.
LacZ transduced neurons were not detected by the α-human α9βl antibody on any substrate thus showing that the antibody binds specifically to its epitope. Rarely, single neurons infected with LacZ-adenoviruses were observed that attached to TN-C or plastic substratum but they did not extend any processes indicating that LacZ transduced adult sensory neurons do not adhere to TN-C and plastic and also do not extend neurites on these substrates.
Similarly to LacZ expressing neurons, α9/α4-adenovirus treated cells did not grow on TN-C or plastic nor did they stain positive for human α9βl when cultured on laminin, indicating that the α9/α4-adenovirus failed to successfully transduce adult sensory neurons. In contrast, α9/α9 and α9/oc5 infected neurons cultured on laminin were labelled with the α-human α9βl antibody and neurites extended on TN-C and tissue culture plastic were all stained too. Moreover, the punctuate staining of axons reveals
that α9βl integrin was localised in clusters, which usually indicates activation of integrin heterodimers .
The number of neurons analysed for human α9 integrin expression on TN-C and plastic was limited and thus it was not possible to determine if there were adherent cells unable to form processes . Neurite outgrowth of transduced neurons on laminin was similar compared to LacZ infected cells. Thus, human α9/α9 and α9/α5 integrin containing adenoviruses successfully transduced adult sensory neurons and ectopic expression of these two human transgenes was detected in cell bodies and neurites of neurons grown on TN-C and tissue culture plastic.
Human α9βl integrin enhances migration of immortalised Schwann cells on the ligand TN-C
Immortalised Schwann cells were transfected with either a construct expressing farnesylated GFP (membrane bound GFP) under the human CMV promoter or with a bicistronic construct expressing human α9βl integrin under the human CMV promoter and farnesylated GFP being under the control of an internal ribosome entry site
(IRES) . Thus, cells transfected with both constructs should be positive for farnesylated GFP. Transfected Schwann cells were plated on to broken cover slips and were allowed to grow until confluency. Cover slips were then inverted on to 3μg/ml mouse TN- C substrate and cells were allowed to migrate for 72hours . Transfected Schwann cells were fixed and labelled for farnesylated GFP. To analyse the migratory behaviour of transfected cells, a grid with each sector measuring lOOμm was used to evaluate the distance of migrating GFP positive Schwann cells from the edge of the broken cover slip.
The transfection efficiency of both constructs was fairly low due to the size of the individual plasmids and was around 20% for the fGFP construct and 5% for the human α9βl integrin IRES fGFP
vector. Whereas fGFP positive cells were brightly labelled with an anti-GFP antibody, staining of human α9βl integrin IRES fGFP producing Schwann cells was often faint, since the fGFP in this construct was under the control of the IRES and translation of the fGFP mRNA was not very efficient .
Control fGFP expressing cells were observed to migrate over the course of 3 days but no further than sector 4 implying that TN-C promotes migration of control Schwann cells to some degree (Fig. 11) . However, human α9βl integrin IRES fGFP positive cells migrated as far as sector 6 and therefore showed an increase in migration compared to only fGFP expressing Schwann cells. Thus, ectopic expression of human α9βl integrin enhanced migration of immortalised Schwann cells on a mouse TN-C substrate.
All transduced PC12 cells adhered significantly better to TN-C and extended neurites to a similar level providing indication that the cytoplasmic domains of human α4, α5, and α9 promote neurite growth and cell adhesion to the same extent. However, in this study, the α9 , α.4 and oc5 cytoplasmic domains had different functional effects in migration assays. Surprisingly, mutants lacking the entire cytoplasmic tail were still able to grow significantly longer neurites on TN-C and plastic than control cells and results were similar to full length α9 integrin, providing indication that α9βl heterodimers without GFFKR are still functional for neurite growth.
Growth of PC12 cells was examined on coronal cryosections of adult brains which provide an environment that is not very growth promoting. The adult CNS expresses low levels of TN-C which could serve as ligand for α9 and α9 chimeric integrins . Neurons were also grown on cortical filter implants which are known to be infiltrated by reactive astrocytes expressing TN-C and inhibitory chondroitin sulphate proteoglycans (CSPGs) . Expression of any of
the α9 integrin constructs enhanced neurite elongation on both cryo frozen brain sections and filter implants. Importantly, these effects were reversed by a α9βl blocking antibody. Thus expression of ectopic human α9 and α9 chimeras enhances neurite growth on inhibitory astrocytic filter membranes. These experiments with PC12 cells show for the first time the role of α9βl integrin in TN-C mediated neurite growth.
In contrast to control LacZ expressing neurons, α9/α9 and α9/α5 positive adult sensory neurons were found to extend processes on TN-C and on plastic, whereas α9/α4 infected cells never formed any neurites . This correlates with the expression levels of α9/α9, α9/α4 and α9/α5 in these neurons. These results provide indication that α9 integrin is developmentally downregulated in sensory neurons with age which correlates with their intrinsic loss to grow on TN-C. Moreover, re-expression of human α9 in adult sensory neurons enhances neurite elongation on TN-C and plastic. Thus, TN-C and EIIIA positive fibronectin are possible neuronal autocrine α9βl ligands that trigger this response.
Schwann cells transplanted into lesioned spinal cords have enhanced regenerative growth of severed axons by promoting elongation of neuronal processes from the spinal cord host tissue into the Schwann cell graft (Barakat et al . , 2005; Pearse and Barakat, 2006) . However, Schwann cell grafts and also endogenous Schwann cells which migrate into the lesioned area would strongly benefit and contribute more to functional recovery, if their capacity to migrate could be enhanced. Here, we propose a role of human α9βl integrin in promoting migration of Schwann cells on the extracellular matrix molecule TN-C.
Schwann cells express a number of integrin heterodimers, some of which are receptors for laminin, fibronectin, vitronectin, and also TN-C. The TN-C receptors α2βl and αvβ3 integrin are reported
to be produced by primary Schwann cells (Milner et al . , 1997) . In particular, αvβ3 integrin is an integrin heterodimer that has been shown to be involved in promoting cell migration on various substrates . The presence of these receptors may explain why Schwann cells expressing only fGFP (control) can migrate on TN-C substrate to some extent .
To date, no function has been described for α9βl integrin in nervous tissue, largely due to the lack of expression (Wang et al . , 1995) . However, in the human immune system, human α9βl integrin has been shown to be expressed on neutrophils and to be involved in migration on TN-C (Taooka et al . , 1999) . The interaction of human α9βl integrin with TN-C has been narrowed down to the peptide AEIDGIEL which is located in the third fibronectin type III repeat and a functional blocking antibody can neutralise the action of human α9βl integrin (Yokosaki et al . , 1998) . Indeed, we could demonstrate that ectopic expression of human α9βl integrin in immortalised Schwann cells enhanced cell motility.
Schwann cells grafted into adult, lesioned spinal cord have been shown to enable severed fibres to grow into Schwann cell bridges, however regenerating fibres often fail to grow back into host CNS tissue and hence the extent of regeneration is limited to the size of the Schwann cell graft (Bunge and Pearse, 2003) . In a therapeutic approach, ectopic expression of human α9βl integrin in transplanted Schwann cells may provoke Schwann cells to migrate out of the lesion site along lesioned tracts and help regenerating fibres to grow for longer distances resulting in improved functional recovery.
Recently, an increased importance has been attributed to Schwann cells produced by the host nervous system contributing to Schwann cell grafts (Hill et al . , 2006) . Although many of the grafted
Schwann cells die, they recruit host Schwann cells before they die (Hill et al . , 2006) . However, these host Schwann cells also become walled off by CNS glia and are unable to migrate along lesioned tracts. In a therapeutical approach, ectopic expression of human α9βl integrin in endogenous Schwann cells may enhance migration first, into adult CNS tissue and second, into a spinal cord lesion. An increase of endogenous Schwann cells at the site of injury would result into an increased number of cells at the site of injury which survive in a hostile and inflammatory environment and subsequently to an increased number of axons growing into the Schwann cell bridge.
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