Neurite growth regulatory factors, antibodies thereto, and pharmaceutical compositions

The CNS myelin associated proteins inhibit neurite outgrowth in nerve cells and neuroblastoma cells, and can also inhibit fibroblast spreading. Such inhibitory proteins include a 35,000 dalton and a 250,000 dalton molecular weight protein and analogs, derivatives, and fragments thereof. The CNS myelin associated inhibitory proteins may be used in the treatment of malignant tumors. The present invention is also directed to antibodies to the CNS myelin associated proteins; such antibodies can be used in the diagnosis and therapies of nerve damage resulting from trauma, infarction, and degenerative disorders of the central nervous system. In a specific embodiment of the invention, monoclonal antibody IN-1 may be used to promote regeneration of nerve fibers over long distances in spinal cord lesions.

TABLE OF CONTENTS 
1. Introduction 
2. Background of the Invention 
2.1. Factors Influencing Neurite Growth 
2.2. Proteases and Their Inhibitors 
2.3. Neuroblastoma 
2.4. Glioblastoma 
3. Summary of the Invention 
3.1. Definitions 
4. Description of the Figures 
5. Detailed Description of the Invention 
5.1. Isolation and Purification of Neurite Growth Regulatory Factors 
5.1.1. Isolation and Purification of CNS Myelin Associated Inhibitory 
Proteins 
5.1.2. Isolation and Purification of Receptors for the CNS Myelin 
Associated Inhibitory Proteins 
5.1.3. Isolation and Purification of Metalloproteases Associated With 
Malignant Tumors 
5.2. Protein Characterization 
5.3. Molecular Cloning of Genes or Gene Fragments Encoding Neurite Growth 
Regulatory Factors 
5.3.1. Isolation and Cloning of the Neurite Growth Regulatory Factor Genes 
5.3.2. Expression of the Cloned Neurite Growth Regulatory Factor Genes 
5.3.3. Identification and Purification of the Expressed Gene Product 
5.3.4. Characterization of the Neurite Growth Regulatory Factor Genes 
5.4. Production of Antibodies to Neurite Growth Regulatory Factors 
5.5. Neurite Growth Regulatory Factor-Related Derivatives, Analogs, and 
Peptides 
5.6. Uses of Neurite Growth Regulatory Factors 
5.6.1. Diagnostic Uses 
5.6.1.1. CNS Myelin Associated Inhibitory Proteins 
5.6.1.2. CNS Myelin Associated Inhibitory Protein Receptors 
5.6.1.3. Metalloproteases and their Inhibitors 
5.6.2. Therapeutic Uses 
5.6.2.1. CNS Myelin Associated Inhibitory Proteins 
5.6.2.2. CNS Myelin Associated Inhibitory Protein Receptors 
5.6.2.3. Metalloproteases and their Inhibitors 
6. Oligodendrocytes and CNS Myelin are Nonpermissive Substrates for Neurite 
Growth and Fibroblast Spreading in Vitro 
6.1. Materials and Methods 
6.1.1. Glial Cell Cultures 
6.1.2. Glia-Nerve Cell Co-Cultures 
6.1.3. Immunofluorescence 
6.1.4. Evaluation of Co-Cultures With Nerve Cells, Neuroblastoma Cells, or 
3T3 Cells 
6.1.5. Preparation of Myelin 
6.2. Results 
6.2.1. Cultures of Dissociated Young or Adult Rat Optic Nerves 
6.2.2. Subtypes of Oligodendrocytes 
6.2.3. Response of Various Cell Types to Highly Branched Oligodendrocytes 
6.2.3.1. Co-Cultures With Sympathetic or Sensory Neurons 
6.2.3.2. Co-Cultures With Fetal Rat Retinal Cells 
6.2.3.3. Response Of Other Cell Types To Highly Branched Oligodendrocytes 
6.2.4. Absence of Species Specificity 
6.2.5. Myelin as a Substrate 
6.3. Discussion 
7. Two Membrane Protein Fractions From Rat Central Nervous System Myelin 
with Inhibitory Properties for Neurite Growth and Fibroblast Spreading 
7.1. Materials and Methods 
7.1.1. Cell Culture 
7.1.2. Sources of Tested Substrates 
7.1.3. Substrate Assaying Procedure 
7.1.4. Substrate-Processing 
7.1.5. Liposomes 
7.1.6. Gel-Extracted Protein Fractions As Substrate 
7.2. Results 
7.2.1. Nonpermissive Substrate Effect is found in CNS Myelin of Higher 
Vertebrates (Chick, Rat), but not Of Lower Vertebrates (Trout, Frog) 
7.2.2. Membrane-Bound Protein Fraction of Rat CNS Myelin is Responsible for 
its Nonpermissive Substrate Properties 
7.2.3. Identification of 35 kD and 250 kD Minor Proteins from Myelin as 
Nonpermissive Substrates For Fibroblast Spreading and Neurite Outgrowth 
7.2.4. Nonpermissive Substrate Property is Enriched in CNS White Matter and 
in Cultured Oligodendrocytes 
7.3. Discussion 
8. Antibody Against Myelin-Associated Inhibitor of Neurite Growth 
Neutralizes Nonpermissive Substrate Properties of CNS White Matter 
8.1. Experimental Procedures 
8.1.1. Cell Culture 
8.1.2. Substrate Preparation 
8.1.3. Immunological Methods 
8.1.3.1. Radioimmunoassay 
8.1.3.2. Immunoblots 
8.1.4. Substrate Testing Procedures 
8.1.5. Neurite Growth Into Optic Nerve Explants In Vitro 
8.2. Results 
8.2.1. Antiserum Against Myelin Neutralizes the Nonpermissive Substrate 
Effects of CNS Myelin and of HBOs 
8.2.2. IN-1: A Monoclonal Antibody Against Gel Purified 250 kD Inhibitor 
from CNS Myelin Neutralizes Myelin Nonpermissiveness 
8.2.3. 250 kD and 35 kD Inhibitors from CNS Myelin Share Two Neutralizing 
Epitopes 
8.2.4. IN-1 Specifically Immunoprecipitates Nonpermissive Substrate 
Activity from Solubilized Myelin Protein 
8.2.5. Nonpermissiveness of Adult Optic Nerve is Neutralized by Absorption 
With IN-1 Antibody 
8.3. Discussion 
9. Involvement of a Metalloprotease in Glioblastoma Infiltration Into 
Central Nervous System Tissue In Vitro 
9.1. Materials and Methods 
9.1.1. Cell Cultures 
9.1.2. Preparation of Nerve Explants for Infiltration Assay 
9.1.3. CNS Frozen Sections and Myelin as Substrates 
9.1.4. C6 Plasma Membranes and Conditioned Medium Preparation 
9.1.5. Treatment of CNS Myelin With C6 Plasma Membranes 
9.2. Results 
9.2.1. C6 Glioblastomas But Not 3T3 Fibroblasts Or B16 Melanomas Infiltrate 
Optic Nerve and CNS White Matter In Vitro 
9.2.2. Glioblastoma Cell Spreading is not Inhibited by CNS Myelin 
9.2.3. Specific Blockers of Metalloproteases Inhibit C6 Cell Spreading on 
CNS Myelin 
9.2.4. A C6 Plasma Membrane-Associated Activity Neutralizes The Inhibitory 
Substrate Property of CNS Myelin 
9.2.5. Inhibitors of Metalloproteases Impair C6 Cell Spreading on CNS White 
Matter and C6 Infiltration of CNS Explants 
9.3. Discussion 
10. Long Distance Tract Regeneration in the Lesioned Spinal Cord of Rats by 
a Monoclonal Antibody Against Myelin-Associated Neurite Growth Inhibitors 
10.1. Materials and Methods 
10.1.1. Pre-Operative Preparation of Animals, Including Implantation of 
Hybridoma Cells 
10.1.2. Procedure for Performing Spinal Cord Lesion 
10.1.3. Post-Lesion Evaluation 
10.2. Results: Regeneration of Corticospinal Tract (CST) Fibers Over Long 
Distances In Rats Bearing IN-1 Secreting Tumors 
10.3. Discussion 
11. Deposit of Hybridomas 
1. INTRODUCTION 
The present invention is directed to genes and their encoded proteins which 
regulate neurite growth, antibodies thereto, and the therapeutic and 
diagnostic uses of such proteins and antibodies. The proteins of the 
present invention include central nervous system myelin associated 
inhibitory proteins, and metalloproteases associated with malignant 
tumors, in particular, primary brain tumors such as glioblastoma and other 
tumors capable of metastasizing to and spreading in the brain. The central 
nervous system myelin associated inhibitory proteins inhibit neurite 
outgrowth and fibroblast spreading and can have important uses in the 
treatment of malignant tumors. Antibodies to such inhibitory proteins can 
have uses in the diagnosis of malignant tumors and in the treatment of 
central nervous system damage and degenerative nerve diseases. In a 
specific embodiment of the invention, antibody to neurite growth inhibitor 
may be used to promote the regeneration of neurons over long distances 
following spinal cord damage. The metalloproteases of the invention allow 
invasive growth of glioblastomas and allow neurite outgrowth in central 
nervous system tissue. They may have important uses in the treatment of 
central nervous system damage and degenerative nerve diseases. Inhibition 
of the metalloprotease can be therapeutically useful in the treatment of 
malignant tumors. 
2. BACKGROUND OF THE INVENTION 
2.1. Factors Influencing Neurite Growth in the Central Nervous System 
Cell attachment, cell spreading, cell motility, and, in particular, neurite 
outgrowth are strongly dependent on cell-substrate interactions (Sanes, 
1983, Ann. Rev. Physiol. 45:581-600; Carbonetto et al., 1987, J. Neurosci. 
7:610-620). An increasing number of substrate molecules favoring 
neuroblast migration or neurite outgrowth have been found in central and 
peripheral nervous tissue (Cornbrooks et al., 1983, Proc. Natl. Acad. Sci. 
USA 80:3850-3854; Edelman, 1984, Exp. Cell Res. 161:1-16; Liesi, 1985, 
EMBO J. 4:1163-1170; Chiu, A. Y. et al., 1986, J. Cell Biol. 
103:1383-1398; Fischer et al., 1986, J. Neurosci. 6:605-612; Lindner et 
al., 1986, Brain Res. 377:298-304; Mirsky et al., 1986, J. Neurocytol. 
15:799-815; Stallcup et al., 1986, J. Neurosci. 5:1090-1101; Carbonetto et 
al., 1987, J. Neurosci. 7:610-620). The appearance of some of these 
factors can be correlated with specific developmental stages, and, in the 
peripheral nervous system (PNS), also with denervation (Edelman, 1984, 
Exp. Cell Res. 161:1-16; Liesi, 1985, EMBO J. 4:1163-1170; Stallcup et 
al., 1985, J. Neurosci. 5:1090-1101; Daniloff et al., 1986, J. Cell Biol. 
103:929-945; Carbonetto et al., 1987, J. Neurosci. 7:610-620). The 
extracellular matrix protein tenascin has been shown to possess 
nonpermissive substrate properties (Chiquet-Ehrismann et al., 1986, Cell 
47:131-139). 
One of the most characterized of the soluble factors favoring neurite 
outgrowth is nerve growth factor (NGF). NGF promotes nerve fiber outgrowth 
from embryonic sensory and sympathetic ganglia in vivo and in vitro as 
well as neurite outgrowth (reviewed in Thoenen et al., 1982, In: Repair 
and Regeneration of the Nervous System, J. G. Nicholls, ed., 
Springer-Verlag, N.Y., pp. 173-185). NGF may also guide the direction of 
such neurite outgrowth. Three different molecular forms of NGF have been 
recognized. One type is a dimer (molecular weight .about.26,000) composed 
of two noncovalently linked, identical polypeptide chains. The second form 
is stable at neutral pH and contains three different polypeptide chains, 
.alpha., .beta. and .gamma. (molecular weight .about.140,000). The .beta. 
chain is the biologically active chain and is identical to the first form 
of NGF. The third form, which is isolated primarily from mouse L cells, 
(see U.S. Pat. No. 4,230,691, by Young, issued Oct. 28, 1980, and 
references therein) has a molecular weight of about 160,000 but is 
unstable at neutral pH. NGF has thus far been isolated from the 
submandibullar glands of mice, mouse L cells, and the prostate gland of 
the guinea pig and bull (reviewed in Thoenen et al., 1982, supra). No 
differences between the biological action of mouse, guinea pig and bull 
NGF have been detected. In addition, NGF isolated from mice have been 
found to bind to the human NGF receptor (Johnson et al., 1986, Cell 
47:545-554). 
The differentiated central nervous system (CNS) of higher vertebrates is 
capable of only very limited regenerative neurite growth after lesions. 
Limited regeneration after lesion has been seen in the retina (McConnell 
and Berry, 1982, Brain Res. 241:362-365) and in aminergic unmyelinated 
fiber tracts after chemical (Bjorklund and Stenevi, 1979, Physiol. Rev. 
59:62-95) but not mechanical lesions (Bregman, 1987, Der. Brain Res. 
34:265-279). Neurite growth from implanted embryonic CNS tissues in adult 
rat CNS has been found in some cases to reach up to 14 mm within some gray 
matter areas, but has not been found to exceed 1 mm within white matter 
(Nornes et al., 1983, Cell Tissue Res. 230:15-35; Bjorklund and Stenevi, 
1979, Physiol. Rev. 59:62-95; Commission, 1984, Neuroscience 12:839-853). 
On the other hand, extensive regenerative growth has been found in the CNS 
of lower vertebrates and in the peripheral nervous system of all 
vertebrates including man. Results from transplantation experiments 
indicate that the lack of regeneration is not an intrinsic property of CNS 
neurons, as these readily extend processes into implanted peripheral 
nervous tissue (Benfey and Aguayo, 1982, Nature (London) 296:150-152; 
Richardson et al., 1984, J. Neurocytol. 13:165-182 and So and Aguayo, 
1985, Brain Res. 328:349-354). PNS neurons, however, failed to extend 
processes into CNS tissue, thus indicating the existence of fundamental 
differences between the two tissues (Aguayo et al., 1978, Neurosci. Lett. 
9:97-104; Weinberg and Spencer, 1979, Brain Res. 162:273-279). 
One major difference between PNS and CNS tissue is the differential 
distribution of the neurite outgrowth promoting extracellular matrix 
component laminin (Liesi, 1985, EMBO J. 4:2505-2511; Carbonetto et al., 
1987, J. Neurosci. 7:610-620). Other factors though may be involved. 
Drastic differences have been observed in neurite growth Supporting 
properties of sciatic and of optic nerve explants in vitro, in spite of 
the presence of laminin immunoreactivity in both explants (Schwab and 
Thoenen, 1985, J. Neurosci. 5:2415-2423). These experiments were carried 
out in the presence of optimal amounts of neurotrophic factors and 
differences persisted upon freezing of tested substrates. 
It has been suggested that the differentiated CNS may lack cellular or 
substrate constituents that are conducive for neurite growth during 
development (Liesi, 1985, EMBO J. 4:2505-2511; and Carbonetto et al, 1987, 
J. Neurosci. 7:610-620), or it may contain components which are 
nonpermissive or inhibitory for nerve fiber regeneration (Schwab and 
Thoenen, 1985, J. Neurosci. 5:2415-2423). 
Recently, a growth (cell proliferation) inhibitory factor for mouse 
neuroblastoma cells was partially purified and characterized from the 
culture medium of fetal rat glioblasts as well as from C6 rat glioma cells 
(Sakazaki et al., 1983, Brain Res. 262:125-135). The factor was estimated 
to have a molecular weight of about 75,000 by gel filtration with BioGel 
P-20 with an isoelectric point of 5.8. The factor did not appear to alter 
the growth rate or morphology of glial cells (C6) or fibroblasts (3T3). In 
addition, no significant nerve growth inhibitory factor activity was 
detected towards neuroblastoma cells (Neuro La, NS-20Y and NIE-115) or 
cloned fibroblasts (3T3). 
2.2. Proteases and Their Inhibitors 
Different proteolytic activities have in the past been shown to be 
increased in tumorigenic cell lines (Matrisian et al., 1986, Proc. Natl. 
Acad. Sci. U.S.A. 83:9413-9417; Mignatti et al., 1986, Cell 47:487-498), 
in primary tumor explants (Mullins and Rohrlich, 1983, Biochem. Biophys. 
Acta 695:177-214), or in transformed cells (Quigley, 1976, J. Cell Biol. 
71:472-486; Mahdavi and Hynes, 1979, Biochem. Biophys. Acta 583:167-178; 
Chen et al., 1984, J. Cell Biol. 98:1546-1555; Wilhelm et al., 1987, Proc. 
Natl. Acad. Sci. U.S.A. 84: 6725-6729). One such group of proteases, 
metalloproteases has been shown to be involved in a number of membrane 
events, including myoblast fusion (Couch and Stritmatter, 1983, Cell 
32:256-265), and exocytosis in mast cells (Mundy and Stritmatter, 1985, 
Cell 40:645-656). 
The isolation and characterization of a plasma membrane-bound 
metalloprotease (endopeptidase 24.11, enkephalinase) was reported by 
Almenoff and Orlowski (1983, Biochemistry 22:590-599). A metalloprotease 
expressed by Rous sarcoma virus transformed chick embryo fibroblasts which 
degrades fibronectin and which was localized at adhesion sites and on 
"invadopodia" was described by Chen and Chen (1987, Cell 48:193-203). 
Studies indicate that proteases and their inhibitors can influence neurite 
extension in neuroblastoma cells (Monard et al., 1983, Prog. Brain Res. 
58:359-363) and in cultured neonatal mouse sensory ganglia (Hawkins and 
Seeds, 1986, Brain Res. 398:63-70). Cultured glial cells and gliomas were 
found to release a 43 kD protein, a glia derived neurite promoting factor 
(GdNPF), which induces neurite outgrowth in neuroblastoma cells but 
inhibits cell migration (Monard, et al., 1983, supra). GdNPF was shown to 
be a very potent inhibitor of cell surface associated serine protease 
activity. Neurite outgrowth from normal mouse sensory ganglia can be 
enhanced by the addition of serine protease inhibitors, ovomucoid trypsin 
inhibitor, leupeptin, soybean trypsin inhibitor, or thrombin (Hawkins and 
Seeds, 1986, supra). In contrast, proteases were found to inhibit such 
neurite outgrowth. Results from preliminary studies indicate that such 
proteases possess a thrombin or trypsin like activity (Hawkins and Seeds, 
1986, supra). 
Other proteases have also been characterized though their functional role 
in neurite outgrowth is as yet unknown. These include a urokinase-like 
plasminogen activator and a calcium dependent metalloprotease released by 
sympathetic and sensory rat neurons (Pittman, 1985, Dev. Biol. 
110:911-101). The metalloprotease was found to have a molecular weight of 
62 kD, to require 1 mM Ca.sup.2+ for calcium activity, and to degrade 
native and denatured collagen more readily than casein, albumin, or 
fibronectin. The plasminogen activator was found to have a molecular 
weight of 51 kD, and was precipitated by a rabbit antiserum produced 
against human urokinase. It may be converted to its active form of 32 kD. 
2.3. Neuroblastoma 
Neuroblastoma arises from neuroectoderm and contains anaplastic sympathetic 
ganglion cells (reviewed in Pinkel and Howarth, 1985, In: Medical 
Oncology, Calabrese, P., Rosenberg, S. A., and Schein, P. S., eds., 
MacMillan, N.Y., pp. 1226-1257). One interesting aspect of neuroblastoma 
is that it has one of the highest rates of spontaneous regression among 
human tumors (Everson, 1964, Ann. NY Acad. Sci. 114:721-735) and a 
correlation exists between such regression and maturation of benign 
ganglioneuroma (Bolande, 1977, Am. J. Dis. Child. 122:12-14). 
Neuroblastoma cells have been found to retain the capacity for 
morphological maturation in culture. The tumors may occur anywhere along 
the sympathetic chain, with 50% of such tumors originating in the adrenal 
medulla. 
Neuroblastoma affects predominantly preschool aged children and is the most 
common extracranial solid tumor in childhood, constituting 6.5% of 
pediatric neoplasms. One half are less than two years of age upon 
diagnosis. Metastases are evident in 60% of the patients at presentation 
usually involving the bones, bone marrow, liver, or skin. The presenting 
symptoms may be related to the primary tumor (spinal coral compression, 
abdominal mass), metastatic tumor (bone pain) or metabolic effects of 
substances such as catecholamines or vasoactive polypeptides secreted by 
the tumor (e.g. hypertension, diarrhea). 
Experimental evidence indicates that an altered response to NGF is 
associated with neuroblastoma (Sonnenfeld and Ishii, 1982, J. Neurosci. 
Res. 8:375-391). NGF stimulated neurite outgrowth in one-half of the 
neuroblastoma cell lines tested; the other half was insensitive. However, 
NGF neither reduced the growth rate nor enhanced survival in any 
neuroblastoma cell line. 
Present therapies for neuroblastoma involve surgery and/or chemotherapy. 
Radiation therapy is used for incomplete tumor responses to chemotherapy. 
There is a 70-100% survival rate in individuals with localized tumors, but 
only a 20% survival rate in those with metastatic disease even with 
multiagent chemotherapy. It appears that patients less than one year have 
a better prognosis (70%) than older children. 
2.4. Glioblastoma 
Glioblastoma is a highly malignant astrocytic tumor usually located in the 
cerebral hemisphere. Astrocytes appear to be a supporting tissue for 
neurons and comprise the vast majority of the intraparenchymal cells of 
the brain (reviewed in Cutler, 1987, In: Scientific American Medicine V. 
2, Rubenstein and Federman, eds., Scientific American, Inc., NY, pp. 1-7). 
Results from a survey conducted by the National Institute of Neurological 
and Communicative Disorders and Stroke indicated that the incidence of 
primary brain tumors in the United States is approximately eight per 
100,000, in which 20% of those tumors are glioblastomas. These tumors are 
generally found in individuals between 45 and 55 years of age. The tumors 
may also involve multiple lobes and may rupture into the ventricular 
system or extend across the corpus collosum to the opposite hemisphere. 
Due to the resulting increase in intracranial pressure, symptoms of tumor 
growth include headache, nausea and vomiting, mental status changes, and 
disturbances of consciousness. Due to their highly invasive properties, 
glioblastomas are associated with a poor prognosis. Chemotherapeutic 
agents or radiotherapies may be used. However, patients generally do not 
survive longer than two years even With these therapies. 
3. SUMMARY OF THE INVENTION 
The present invention relates to genes and their encoded proteins which 
regulate neurite growth and the diagnostic and therapeutic uses of such 
proteins. Such proteins are termed herein neurite growth regulatory 
factors. The neurite growth regulatory factors of the present invention 
include, in one embodiment, central nervous system myelin associated 
proteins which inhibit neurite outgrowth, and are termed herein neurite 
growth inhibitory factors. Another embodiment of the invention is directed 
to neurite growth regulatory factors which are metalloproteases associated 
with malignant tumors, in particular, those tumors metastatic to the 
brain. Such metalloproteases enable the malignant cells to overcome the 
inhibitory CNS environment and invade large areas of brain and spinal 
cord. 
The CNS myelin associated proteins inhibit neurite outgrowth in nerve cells 
and neuroblastoma cells and also inhibit the spreading of fibroblasts and 
melanoma cells. Such inhibitory proteins include but are not limited to 
35,000 dalton and a 250,000 dalton molecular weight proteins and analogs, 
derivatives, and fragments thereof. The CNS myelin associated inhibitory 
proteins may be used in the treatment of patients with malignant tumors 
which include but are not limited to melanoma and nerve tissue tumors 
(e.g., neuroblastoma). The absence of the myelin associated inhibitory 
proteins can be diagnostic for the presence of a malignant tumor such as 
those metastatic to the brain (e.g., glioblastoma). The present invention 
also relates to antagonists of the CNS myelin associated inhibitory 
proteins, including, but not limited to, antibodies, i.e. antibodies IN-1 
or IN-2. Such antibodies can be used to neutralize the neurite growth 
inhibitory factors for regenerative repair after trauma, degeneration., or 
inflammation. In a further specific embodiment, monoclonal antibody IN-1 
may be used to promote regeneration of nerve fibers over long distances 
following spinal cord damage. 
The present invention further relates to neurite growth regulatory factor 
receptors and fragments thereof as well as the nucleic acid sequences 
coding for such neurite growth regulatory factor receptors and fragments, 
and their therapeutic and diagnostic uses. Substances which function as 
either agonists or antagonists to neurite growth regulatory factor 
receptors are also envisioned and within the scope of the present 
invention. 
The metalloproteases of the present invention can be found associated with 
malignant tumors, in particular, those capable of metastasizing to the 
brain. In a specific embodiment, the metalloprotease is associated with 
membranes of glioblastoma cells. The metalloproteases, and analogs, 
derivatives, and fragments thereof can have value in the treatment of 
nerve damage resulting from trauma, stroke, degenerative disorders of the 
central nervous system, etc. In another embodiment of the invention, the 
metalloprotease may be used in combination with antibodies to the neurite 
growth inhibitory factors to treat nerve damage. 
The present invention is also directed to inhibitors of and/or antibodies 
to the metalloproteases of the invention. Such inhibitors and/or 
antibodies can be used in the diagnosis and/or treatment of malignant 
tumors such as those which can metastasize to the brain, including but not 
limited to glioblastomas. Alternatively, the metalloprotease inhibitors, 
in combination with CNS myelin associated inhibitory protein or analogs, 
derivatives, or fragments thereof, may be used in the treatment and/or 
diagnosis of malignant tumors including but not limited to glioblastoma, 
neuroblastoma, and melanoma. 
3.1. Definitions 
As used herein, the following terms shall have the meanings indicated: 
BSA: bovine serum albumin 
cbz-tyr-tyr: carbobenzoxy-tryosine-tyrosine 
cbz-gly-phe-NH.sub.2 : carbobenzoxy-glycine-phenylalanine-amide 
cbz-ala-phe-NH.sub.2 : carbobenzoxy-alanine-phenylalanine-amide 
cbz-phe-phe-NH.sub.2 : carbobenzoxy-phenylalanine-phenylalanine-amide 
cbz-gly-phe-phe-NH.sub.2 : 
carbobenzoxy-glycine-phenylalanine-phenylalanine-amide 
CNS: central nervous system 
CST: Corticospinal tract 
DMEM: Dulbecco's Modified Minimal Essential Media 
EDTA: ethylenediamine tetracetate 
EGTA: ethylene glycol-bis-(.beta.-aminoethyl ether)-N,N,N'-N'-tetracetate 
FCS: fetal calf serum 
FITC: fluorescein isothiocyanate 
GdNPF: glial-derived neurite promoting factor 
GFAP: glial fibrillary acid protein 
HBO: highly branched oligodendrocyte 
Hepes: N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid 
IN-1: a monoclonal antibody against gel-purified 250 kD CNS myelin 
associated inhibitory protein 
IN-2: a monclonal antibody against gel-purified 35 kD CNS myelin associated 
inhibitory protein 
J1: a cell adhesion molecule of molecular weight 160-180 kD 
kD: kilodalton 
Mab: monoclonal antibody 
MW: molecular weight 
N-CAM: neural cell adhesion molecule 
NGF: nerve growth factor 
neurite growth 
regulatory factors: CNS myelin associated 35 kD and 250 kD inhibitory 
proteins, and a glioblastoma cell membrane associated metalloprotease 
PBS: phosphate buffered saline 
PLYS: poly-D-lysine 
PNS: peripheral nervous system 
PORN: polyornithine 
SCG: superior cervical ganglion 
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis 
Tris: Tris (hydroxymethyl) aminomethane

5. DETAILED DESCRIPTION OF THE INVENTION 
The present invention is directed to genes and their encoded proteins which 
regulate neurite growth and the diagnostic and therapeutic uses of such 
proteins. The proteins of the present invention (termed herein neurite 
growth regulatory factors) include proteins associated with central 
nervous system myelin with highly nonpermissive substrate properties, 
termed herein neurite growth inhibitory factors. The neurite growth 
regulatory factors also include metalloproteases which can be found 
associated with malignant tumors, in particular, those tumors metastatic 
to the brain. 
The CNS myelin associated proteins of the invention inhibit neurite 
outgrowth in nerve cells or neuroblastoma cells. The protein can also 
inhibit fibroblast spreading and migration. These inhibitory proteins are 
active cross-species and may be used in the treatment of patients with 
malignant tumors including but not limited to melanoma and tumors of nerve 
tissue (e.g. neuroblastoma). In a specific example of the present 
invention, a 35 kilodalton and a 250 kilodalton CNS myelin associated 
protein are described. 
The present invention is also directed to antibodies to the CNS myelin 
associated proteins and their therapeutic and diagnostic uses. These 
antibodies can be used in the treatment of nerve damage resulting from, 
e.g., trauma (e.g., spinal cord injuries), stroke, degenerative disorders 
of the central nervous system, etc. In particular, antibodies to CNS 
myelin associated proteins may be used to promote regeneration of nerve 
fibers. In a specific embodiment of the invention, monoclonal antibody 
IN-1 may be used to promote the regeneration of nerve fibers over long 
distances following spinal cord damage. 
The present invention further relates to neurite growth regulatory factor 
receptors and peptide fragments thereof as well as the nucleic acid 
sequences coding for neurite growth regulatory factor receptors and 
fragments, and their therapeutic and diagnostic uses. Antibodies to 
neurite growth regulatory factor receptors are also envisioned and within 
the scope of the present invention. 
The present invention is also directed to metalloproteases associated with 
malignant tumors, in particular, those metastatic to the brain. In a 
specific embodiment, the metalloprotease is associated with glioblastoma 
cells. The metalloproteases of the invention are associated with the CNS 
infiltration activity of malignant cells, and can neutralize the 
inhibitory substrate properties of the CNS myelin-associated proteins. The 
metalloproteases can have therapeutic value in the treatment of nerve 
damage such as that resulting from traumatic injury (e.g. spinal cord 
injuries), stroke, degenerative disorders of the central nervous system, 
etc. Alternatively, the metalloprotease may be used in combination with 
antibodies directed against myelin associated inhibitory proteins to treat 
nerve damage. 
The present invention is also directed to inhibitors of the 
metalloproteases. Such inhibitors can impair malignant cell spreading and 
infiltration, and can be used in the treatment of malignant tumors (e.g. 
glioblastoma). In a specific embodiment, the metalloprotease inhibitors in 
combination with CNS myelin associated inhibitory proteins such as the 
35,000 dalton and/or the 250,000 dalton molecular weight proteins or human 
functional equivalents thereof, may be used in the diagnosis and/or 
treatment of malignant tumors which include but are not limited to 
glioblastomas, neuroblastomas, and melanomas. 
The method of the invention can be divided into the following stages, 
solely for the purpose of description: (1) isolation and purification of 
neurite growth regulatory factors; (2) characterization of neurite growth 
regulatory factors; (3) molecular cloning of genes or gene fragment 
encoding neurite growth regulatory factors; (4) production of antibodies 
against neurite growth regulatory factors; and (5) generation of neurite 
growth regulatory factor related derivatives, analogs, and peptides. The 
method further encompasses the diagnostic and therapeutic uses of neurite 
growth regulatory factors and their antibodies. 
5.1. Isolation and Purification of Neurite Growth Regulatory Factors 
The present invention relates to CNS myelin associated inhibitory proteins 
of neurite growth, receptors of CNS myelin associated inhibitory proteins 
of neurite growth, and to metalloproteases such as that associated with 
membranes of glioblastoma cells. The CNS myelin associated inhibitory 
proteins of the invention may be isolated by first isolating myelin and 
subsequent purification therefrom. Similarly, the metalloprotease may be 
obtained by isolation from mammalian glioblastoma cells. Isolation 
procedures which may be employed are described more fully in the sections 
which follow. Alternatively, the CNS myelin associated inhibitory proteins 
or the metalloprotease may be obtained from a recombinant expression 
system (see Section 5.3., infra). Procedures for the isolation and 
purification of receptors for the CNS myelin associated inhibitory 
proteins are described in Section 5.1.2., infra. 
5.1.1. Isolation and Purification of CNS Myelin Associated Inhibitory 
Proteins 
CNS myelin associated inhibitory proteins can be isolated from the CNS 
myelin of higher vertebrates including, but not limited to, birds or 
mammals. Myelin can 30 be obtained from the optic nerve or from central 
nervous system tissue that includes but is not limited to spinal cords or 
brain stems. The tissue may be homogenized using procedures described in 
the art (Colman et al., 1982, J. Cell Biol. 95:598-608). The myelin 
fraction can be isolated subsequently also using procedures described 
(Colman et al., 1982, supra). 
In one embodiment of the invention, the CNS myelin associated inhibitory 
proteins can be solubilized in detergent (e.g., Nonidet P-40.TM., sodium 
deoxycholate). The solubilized proteins can subsequently be purified by 
various procedures known in the art, including but not limited to 
chromatography (e.g., ion exchange, affinity, and sizing chromatography), 
centrifugation, electrophoretic procedures, differential solubility, or by 
any other standard technique for the purification of proteins (see, e.g., 
Section 7.2.3., infra). 
Alternatively, the CNS myelin associated inhibitory proteins may be 
isolated and purified using immunological procedures. For example, in one 
embodiment of the invention, the proteins can first be solubilized using 
detergent (e.g., Nonidet P-40.TM., sodium deoxycholate). The proteins may 
then be isolated by immunoprecipitation with antibodies to the 35 
kilodalton and/or the 250 kilodalton proteins. Alternatively, the CNS 
myelin associated inhibitory proteins may be isolated using immunoaffinity 
chromatography in which the proteins are applied to an antibody column in 
solubilized form. 
5.1.2. Isolation and Purification of Receptors for the CNS Myelin 
Associated Inhibitory Proteins 
Receptors for the CNS myelin associated inhibitory proteins can be isolated 
from cells whose attachment, spreading, growth and/or motility is 
inhibited by the CNS myelin associated inhibitory proteins. Such cells 
include but are not limited to fibroblasts and neurons. In a preferred 
embodiment, fibroblasts are used as the source for isolation and 
purification of the receptors. 
In one embodiment, receptors to CNS myelin associated inhibitory proteins 
may be isolated by affinity chromatography of fibroblast cell extracts, in 
which a myelin associated inhibitory protein or peptide fragment thereof 
is immobilized to a solid support. 
5.1.3. Isolation and Purification of Metalloproteases Associated with 
Malignant Tumors 
The metalloproteases of the present invention may be isolated from cells of 
malignant tumors, in particular, those tumors which can metastasize to the 
brain. In a preferred embodiment, a metalloprotease can be isolated from 
mammalian glioblastoma cells. In a preferred method, the metalloprotease 
is isolated from the plasma membrane fraction of such cells. The cells may 
be obtained by dissociating and homogenizing tumors using procedures known 
in the art or from tumor cell lines. Plasma membrane fractions may be 
obtained using procedures known in the art, e.g., gradient centrifugation 
(Quigley, 1976, J. Cell Biol. 71:472-486). The metalloprotease may be 
isolated from the membranes by solubilization with mild ionic or nonionic 
detergent (e.g., deoxycholate, Nonidet P-40.TM., Triton.TM., Brij.TM.) 
using procedures described in the art (reviewed in Cooper, 1977, In Tools 
of Biochemistry, John Wiley & Sons, N.Y., pp. 355-406). 
Purification of the metalloprotease can be carried out by known procedures, 
including but not limited to chromatography (e.g., ion exchange, affinity, 
and sizing column chromatography), centrifugation, electrophoretic 
procedures, differential solubility, or by any other standard technique 
for the purification of proteins. 
5.2. Protein Characterization 
The neurite growth regulatory factors of the present invention can be 
analyzed by assays based on their physical, immunological, or functional 
properties. The half life of the neurite growth regulatory factors in 
cultured cells can be studied, for example, by use of cycloheximide, an 
inhibitor of protein synthesis (Vasquez, 1974, FEBS Lett. 40:563-584). In 
other experiments, a physiological receptor for a neurite growth 
regulatory factor could be identified by assays which detect complex 
formation with a neurite growth regulatory factor, e.g., by use of 
affinity chromatography with immobilized neurite growth regulatory factor, 
binding to a labeled neurite growth regulatory factor followed by 
cross-linking and immunoprecipitation, etc. 
Electrophoretic techniques such as SDS-polyacrylamide gel electrophoresis 
and two-dimensional electrophoresis can be used to study protein 
structure. Other techniques which can be used include but are not limited 
to peptide mapping, isoelectric focusing, and chromatographic techniques. 
The amino acid sequences of primary myelin associated inhibitors or of the 
metalloprotease can be derived by deduction from the DNA sequence if such 
is available, or alternatively, by direct sequencing of the protein, e.g., 
with an automated amino acid sequencer. The protein sequences can be 
further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, 
Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828). A hydrophilicity profile can 
be used to identify the hydrophobic and hydrophilic regions of the protein 
(and the corresponding regions of the gene sequence, if available, which 
encode such regions). 
Secondary structural analysis (Chou and Fasman, 1974, Biochemistry 13:222) 
can also be done, to identify regions of the CNS myelin associated 
inhibitor or gliobastoma metalloprotease sequence that assume specific 
secondary structures. Other methods of structural analysis can also be 
employed. These include but are not limited to X-ray crystallography 
(Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modeling 
(Fletterick, R. and Zoller, M. (eds.), 1986, Computer Graphics and 
Molecular Modeling, in Current Communications in Molecular Biology, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). 
5.3. Molecular Cloning of Genes or Gene Fragments Encoding Neurite Growth 
Regulatory Factors 
5.3.1. Isolation and Cloning of the Neurite Growth Regulatory Factor Genes 
Any mammalian cell can potentially serve as the nucleic acid source for the 
molecular cloning of the genes encoding the CNS myelin associated 
inhibitory proteins, including but not limited to the 35 kD and/or 250 kD 
myelin associated proteins described in Section 7., infra, or the 
glioblastoma associated metalloprotease, hereinafter referred to as 
neurite growth regulatory factor genes. 
The DNA may be obtained by standard procedures known in the art from cloned 
DNA (e.g., a DNA "library"), by chemical synthesis, by cDNA cloning, or by 
the cloning of genomic DNA, or fragments thereof, purified from the 
desired mammalian cell. (See, for example, Maniatis et al., 1982, 
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 
Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A 
Practical Approach, MRL Press, Ltd., Oxford, U.K., Vol. I, II.) Clones 
derived from genomic DNA may contain regulatory and intron DNA regions, in 
addition to coding regions; clones derived from cDNA will contain only 
exon sequences. Whatever the source, a given neurite growth regulatory 
factor gene should be molecularly cloned into a suitable vector for 
propagation of the gene. 
In the molecular cloning of a neurite growth regulatory factor gene from 
genomic DNA, DNA fragments are generated, some of which will encode the 
desired neurite growth regulatory factor gene. The DNA may be cleaved at 
specific sites using various restriction enzymes. Alternatively, one may 
use DNAse in the presence of manganese to fragment the DNA, or the DNA can 
be physically sheared, as for example, by sonication. The linear DNA 
fragments can then be separated according to size by standard techniques, 
including but not limited to, agarose and polyacrylamide gel 
electrophoresis and column chromatography. 
Once the DNA fragments are generated, identification of the specific DNA 
fragment containing a neurite growth regulatory factor gene may be 
accomplished in a number of ways. For example, if an amount of a neurite 
growth regulatory factor gene or its specific RNA, or a fragment thereof, 
is available and can be purified and labeled, the generated DNA fragments 
may be screened by nucleic acid hybridization to the labeled probe (Benton 
and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. 
Acad. Sci. U.S.A. 72:3961-3965). For example, in a preferred embodiment, a 
portion of a neurite growth regulatory factor amino acid sequence can be 
used to deduce the DNA sequence, which DNA sequence can then be 
synthesized as an oligonucleotide for use as a hybridization probe. 
Alternatively, if a purified neurite growth regulatory factor probe is 
unavailable, nucleic acid fractions enriched in neurite growth regulatory 
factor may be used as a probe, as an initial selection procedure. 
It is also possible to identify an appropriate neurite growth regulatory 
factor-encoding fragment by restriction enzyme digestion(s) and comparison 
of fragment sizes with those expected according to a known restriction map 
if such is available. Further selection on the basis of the properties of 
the gene, or the physical, chemical, or immunological properties of its 
expressed product, as described supra, can be employed after the initial 
selection. 
A neurite growth regulatory factor gene can also be identified by mRNA 
selection using nucleic acid hybridization followed by in vitro 
translation or translation in Xenopus oocytes. In an example of the latter 
procedure, oocytes are injected with total or size fractionated CNS mRNA 
populations, and the membrane-associated translation products are screened 
in a functional assay (3T3 cell spreading). Preadsorption of the RNA with 
complementary DNA (cDNA) pools leading to the absence of expressed 
inhibitory factors indicates the presence of the desired cDNA. Reduction 
of pool size will finally lead to isolation of a single cDNA clone. In an 
alternative procedure, DNA fragments can be used to isolate complementary 
mRNAs by hybridization. Such DNA fragments may represent available, 
purified neurite growth regulatory factor DNA, or DNA that has been 
enriched for neurite growth regulatory factor sequences. 
Immunoprecipitation analysis or functional assays of the in vitro 
translation products of the isolated mRNAs identifies the mRNA and, 
therefore, the cDNA fragments that contain neurite growth regulatory 
factor sequences. An example of such a functional assay involves an assay 
for nonpermissiveness in which the effect of the various translation 
products on the spreading of 3T3 cells on a polylysine coated tissue 
culture dish is observed (see Section 7.1.2., infra). In addition, 
specific mRNAs may be selected by adsorption of polysomes isolated from 
cells to immobilized antibodies specifically directed against a neurite 
growth regulatory factor protein. A radiolabeled neurite growth regulatory 
factor cDNA can be synthesized using the selected mRNA (from the adsorbed 
polysomes) as a template. The radiolabeled mRNA or cDNA may then be used 
as a probe to identify the neurite growth regulatory factor DNA fragments 
from among other genomic DNA fragments. 
Alternatives to isolating the neurite growth regulatory factor genomic DNA 
include, but are not limited to, chemically synthesizing the gene sequence 
itself from a known sequence or making cDNA to the mRNA which encodes the 
neurite growth regulatory factor gene. Other methods are possible and 
within the scope of the invention. 
The identified and isolated gene or cDNA can then be inserted into an 
appropriate cloning vector. A large number of vector-host systems known in 
the art may be used. Possible vectors include, but are not limited to, 
cosmids, plasmids or modified viruses, but the vector system must be 
compatible with the host cell used. Such vectors include, but are not 
limited to, bacteriophages such as lambda derivatives, or plasmids such as 
pBR322 or pUC plasmid derivatives. Recombinant molecules can be introduced 
into host cells via transformation, transfection, infection, 
electroporation, etc. 
In an alternative embodiment, the neurite growth regulatory factor gene may 
be identified and isolated after insertion into a suitable cloning vector, 
in a "shot gun" approach. Enrichment for a given neurite growth regulatory 
factor gene, for example, by size fractionation or subtraction of cDNA 
specific to low neurite growth regulatory factor producers, can be done 
before insertion into the cloning vector. In another embodiment, DNA may 
be inserted into an expression vector system, and the recombinant 
expression vector containing a neurite growth regulatory factor gene may 
then be detected by functional assays for the neurite growth regulatory 
factor protein. 
The neurite growth regulatory factor gene is inserted into a cloning vector 
which can be used to transform, transfect, or infect appropriate host 
cells so that many copies of the gene sequences are generated. This can be 
accomplished by ligating the DNA fragment into a cloning vector which has 
complementary cohesive termini. However, if the complementary restriction 
sites used to fragment the DNA are not present in the cloning vector, the 
ends of the DNA molecules may be enzymatically modified. Alternatively, 
any site desired may be produced by ligating nucleotide sequences 
(linkers) onto the DNA termini; these ligated linkers may comprise 
specific chemically synthesized oligonucleotides encoding restriction 
endonuclease recognition sequences. In an alternative method, the cleaved 
vector and neurite growth regulatory factor gene may be modified by 
homopolymeric tailing. 
Identification of the cloned neurite growth regulatory factor gene can be 
accomplished in a number of ways based on the properties of the DNA 
itself, or alternatively, on the physical, immunological, or functional 
properties of its encoded protein. For example, the DNA itself may be 
detected by plaque or colony nucleic acid hybridization to labeled probes 
(Benton, W. and Davis, R., 1977, Science 196:180; Grunstein, M. and 
Hogness, D., 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Alternatively, 
the presence of a neurite growth regulatory factor gene may be detected by 
assays based on properties of its expressed product. For example, cDNA 
clones, or DNA clones which hybrid-select the proper mRNAs, can be 
selected which produce a protein that inhibits in vitro neurite outgrowth. 
If an antibody to a neurite growth regulatory factor is available, a 
neurite growth regulatory factor protein may be identified by binding of 
labeled antibody to the putatively neurite growth regulatory 
factor-synthesizing clones, in an ELISA (enzyme-linked immunosorbent 
assay)-type procedure. 
In specific embodiments, transformation of host cells with recombinant DNA 
molecules that incorporate an isolated neurite growth regulatory factor 
gene, cDNA, or synthesized DNA sequence enables generation of multiple 
copies of the gene. Thus, the gene may be obtained in large quantities by 
growing transformants, isolating the recombinant DNA molecules from the 
transformants and, when necessary, retrieving the inserted gene from the 
isolated recombinant DNA. 
If the ultimate goal is to insert the gene into virus expression vectors 
such as vaccinia virus or adenovirus, the recombinant DNA molecule that 
incorporates a neurite growth regulatory factor gene can be modified so 
that the gene is flanked by virus sequences that allow for genetic 
recombination in cells infected with the virus so that the gene can be 
inserted into the viral genome. 
After the neurite growth regulatory factor DNA-containing clone has been 
identified, grown, and harvested, its DNA insert may be characterized as 
described in Section 5.3.4, infra. When the genetic structure of a neurite 
growth regulatory factor gene is known, it is possible to manipulate the 
structure for optimal use in the present invention. For example, promoter 
DNA may be ligated 5' of a neurite growth regulatory factor coding 
sequence, in addition to or replacement of the native promoter to provide 
for increased expression of the protein. Many manipulations are possible, 
and within the scope of the present invention. 
5.3.2. Expression of the Cloned Neurite Growth Regulatory Factor Genes 
The nucleotide sequence coding for a neurite growth regulatory factor 
protein or a portion thereof, can be inserted into an appropriate 
expression vector, i.e., a vector which contains the necessary elements 
for the transcription and translation of the inserted protein-coding 
sequence. The necessary transcriptional and translation signals can also 
be 30 supplied by the native neurite growth regulatory factor gene and/or 
its flanking regions. A variety of host-vector systems may be utilized to 
express the protein-coding sequence. These include but are not limited to 
mammalian cell systems infected with virus (e.g., vaccinia virus, 
adenovirus, etc.); insect cell systems infected with virus (e.g., 
baculovirus); microorganisms such as yeast containing yeast vectors, or 
bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. 
The expression elements of these vectors vary in their strengths and 
specificities. Depending on the host-vector system utilized, any one of a 
number of suitable transcription and translation elements may be used. 
Any of the methods previously described for the insertion of DNA fragments 
into a vector may be used to construct expression vectors containing a 
chimeric gene consisting of appropriate transcriptional/translational 
control signals and the protein coding sequences. These methods may 
include in vitro recombinant DNA and synthetic techniques and in vivo 
recombinations (genetic recombination). 
Expression vectors containing neurite growth regulatory factor gene inserts 
can be identified by three general approaches: (a) DNA-DNA hybridization, 
(b) presence or absence of "marker" gene functions, and (c) expression of 
inserted sequences. In the first approach, the presence of a foreign gene 
inserted in an expression vector can be detected by DNA-DNA hybridization 
using probes comprising sequences that are homologous to an inserted 
neurite growth regulatory factor gene. In the second approach, the 
recombinant vector/host system can be identified and selected based upon 
the presence or absence of certain "marker" gene functions (e.g., 
thymidine kinase activity, resistance to antibiotics, transformation 
phenotype, occlusion body formation in baculovirus, etc.) caused by the 
insertion of foreign genes in the vector. For example, if a given neurite 
growth regulatory factor gene is inserted within the marker gene sequence 
of the vector, recombinants containing the neurite growth regulatory 
factor insert can be identified by the absence of the marker gene 
function. In the third approach, recombinant expression vectors can be 
identified by assaying the foreign gene product expressed by the 
recombinant. Such assays can be based on the physical, immunological, or 
functional properties of a given neurite growth regulatory factor gene 
product. 
Once a particular recombinant DNA molecule is identified and isolated, 
several methods known in the art may be used to propagate it. Once a 
suitable host system and growth conditions are established, recombinant 
expression vectors can be propagated and prepared in quantity. As 
previously explained, the expression vectors which can be used include, 
but are not limited to, the following vectors or their derivatives: human 
or animal viruses such as vaccinia virus or adenovirus; insect viruses 
such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), 
and plasmid and cosmid DNA vectors, to name but a few. 
In addition, a host cell strain may be chosen which modulates the 
expression of the inserted sequences, or modifies and processes the gene 
product in the specific fashion desired. Expression from certain promoters 
can be elevated in the presence of certain inducers; thus, expression of 
the genetically engineered neurite growth regulatory factor protein may be 
controlled. Furthermore, different host cells have characteristic and 
specific mechanisms for the translational and post-translational 
processing and modification (e.g., glycosylation, cleavage) of proteins. 
Appropriate cell lines or host systems can be chosen to ensure the desired 
modification and processing of the foreign protein expressed. For example, 
expression in a bacterial system can be used to produce an unglycosylated 
core protein product. Expression in yeast will produce a glycosylated 
product. Expression in mammalian (e.g. COS) cells can be used to ensure 
"native" glycosylation of the heterologous neurite growth regulatory 
factor protein. Furthermore, different vector/host expression systems may 
effect processing reactions such as proteolytic cleavages to different 
extents. 
5.3.3. Identification and Purification of the Expressed Gene Product 
Once a recombinant which expresses a given neurite growth regulatory factor 
gene is identified, the gene product can be purified as described in 
Section 5.1, supra, and analyzed as described in Section 5.2, supra. 
The amino acid sequence of a given neurite growth regulatory factor protein 
can be deduced from the nucleotide sequence of the cloned gene, allowing 
the protein, or a fragment thereof, to be synthesized by standard chemical 
methods known in the art (e.g., see Hunkapiller, et al., 1984, Nature 
310:105-111). 
In particular embodiments of the present invention, such neurite growth 
regulatory factor proteins, whether produced by recombinant DNA techniques 
or by chemical synthetic methods, include but are not limited to those 
containing altered sequences in which functionally equivalent amino acid 
residues are substituted for residues within the sequence resulting in a 
silent change. For example, one or more amino acid residues within the 
sequence can be substituted by another amino acid of a similar polarity 
which acts as a functional equivalent, resulting in a silent alteration. 
Substitutes for an amino acid within the sequence may be selected from 
other members of the class to which the amino acid belongs. For example, 
the nonpolar (hydrophobic) amino acids include alanine, leucine, 
isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. 
The polar neutral amino acids include glycine, serine, threonine, 
cysteine, tyrosine, asparagine, and glutamine. The positively charged 
(basic) amino acids include arginine, lysine, and histidine. The 
negatively charged (acidic) amino acids include aspartic acid and glutamic 
acid. Also included within the scope of the invention are neurite growth 
regulatory factor proteins which are differentially modified during or 
after translation, e.g., by glycosylation, proteolytic cleavage, etc. 
5.3.4. Characterization of the Neurite, Growth Regulatory Factor Genes 
The structure of a given neurite growth regulatory factor gene can be 
analyzed by various methods known in the art. 
The cloned DNA or cDNA corresponding to a given neurite growth regulatory 
factor gene can be analyzed by methods including but not limited to 
Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), 
Northern hybridization (Alwine, et al., 1977, Proc. Natl. Acad. Sci. 
U.S.A. 74:5350-5354; Wahl, et al., 1987, Meth. Enzymol. 152:572-581), 
restriction endonuclease mapping (Maniatis, et al., 1982, Molecular 
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring 
Harbor, N.Y.), and DNA sequence analysis. 
DNA sequence analysis can be performed by any techniques known in the art 
including but not limited to the method of Maxam and Gilbert (1980, Meth. 
Enzymol. 65:499-560), the Sanger dideoxy method (Sanger, et al., 1977, 
Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467), or use of an automated DNA 
sequenator (e.g., Applied Biosystems, Foster City, Calif.). 
5.4. Production of Antibodies to Neurite, Growth Regulatory Factors 
Antibodies can be produced which recognize neurite growth regulatory 
factors or related proteins. Such antibodies can be polyclonal or 
monoclonal. 
Various procedures known in the art may be used for the production of 
polyclonal antibodies to epitopes of a given neurite growth regulatory 
factor. For the production of antibody, various host animals can be 
immunized by injection with a neurite growth regulatory factor protein, or 
a synthetic protein, or fragment thereof, including but not limited to 
rabbits, mice, rats, etc. Various adjuvants may be used to increase the 
immunological response, depending on the host species, and including but 
not limited to Freund's (complete and incomplete), mineral gels such as 
aluminum hydroxide, surface active substances such as lysolecithin, 
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet 
hemocyanins, dinitrophenol, and potentially useful human adjuvants such as 
BCG (bacille Calmette-Guerin) and corynebacterium parvum. 
A monoclonal antibody to an epitope of a neurite growth regulatory factor 
can be prepared by using any technique which provides for the production 
of antibody molecules by continuous cell lines in culture. These include 
but are not limited to the hybridoma technique originally described by 
Kohler and Milstein (1975, Nature 256:495-497), and the more recent human 
B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72) 
and EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and 
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In a particular 
embodiment, the procedure described infra in Section 8.1. may be used to 
obtain mouse monoclonal antibodies which recognize the 35 kD and 250 kD 
CNS myelin associated inhibitory proteins. 
The monoclonal antibodies for therapeutic use may be human monoclonal 
antibodies or chimetic human-mouse (or other species) monoclonal 
antibodies. Human monoclonal antibodies may be made by any of numerous 
techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. 
Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today 4:72-79; 
Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimetic antibody molecules 
may be prepared containing a mouse antigen-binding domain with human 
constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 
81:6851, Takeda et al., 1985, Nature 314:452). 
A molecular clone of an antibody to a neurite growth regulatory factor 
epitope can be prepared by known techniques. Recombinant DNA methodology 
(see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) may be used to 
construct nucleic acid sequences which encode a monoclonal antibody 
molecule, or antigen binding region thereof. 
Antibody molecules may be purified by known techniques, e.g., 
immunoabsorption or immunoaffinity chromatography, chromatographic methods 
such as HPLC (high performance liquid chromatography), or a combination 
thereof, etc. 
Antibody fragments which contain the idiotype of the molecule can be 
generated by known techniques. For example, such fragments include but are 
not limited to: the F(ab').sub.2 fragment which can be produced by pepsin 
digestion of the antibody molecule; the Fab' fragments which can be 
generated by reducing the disulfide bridges of the F(ab').sub.2 fragment, 
and the 2 Fab or Fab fragments which can be generated by treating the 
antibody molecule with papain and a reducing agent. 
5.5. Neurite Growth Regulatory Factor-Related Derivatives, Analogs, and 
Peptides 
The production and use of derivatives, analogs, and peptides related to a 
given neurite growth regulatory factor are also envisioned, and within the 
scope of the present invention and include molecules antagonistic to 
neurite growth. regulatory factors (for example, and not by way of 
limitation, anti-idiotype antibodies). Such derivatives, analogs, or 
peptides which have the desired inhibitory activity can be used, for 
example, in the treatment of neuroblastoma (see Section 5.6, infra). 
Derivatives, analogs, or peptides related to a neurite growth regulatory 
factor can be tested for the desired activity by assays for nonpermissive 
substrate effects. For example, procedures such as the assay for 
nonpermissiveness in which the effect of the various translation products 
on the spreading of 3T3 cells on a polylysine coated tissue culture dish 
is observed (see Section 7.1.2., infra). 
The neurite growth regulatory factor-related derivatives, analogs, and 
peptides of the invention can be produced by various methods known in the 
art. The manipulations which result in their production can occur at the 
gene or protein level. For example, a cloned neurite growth regulatory 
factor gene can be modified by any of numerous strategies known in the art 
(Maniatis, et al., 1982, Molecular Cloning, A Laboratory Manual, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). A given neurite 
growth regulatory factor sequence can be cleaved at appropriate sites with 
restriction endonuclease(s), subjected to enzymatic modifications if 
desired, isolated, and ligated in vitro. In the production of a gene 
encoding a derivative, analogue, or peptide related to a neurite growth 
regulatory factor, care should be taken to ensure that the modified gene 
remains within the same translational reading frame as the neurite growth 
regulatory factor, uninterrupted by translational stop signals, in the 
gene region where the desired neurite growth regulatory factor-specific 
activity is encoded. 
Additionally, a given neurite growth regulatory factor gene can be mutated 
in vitro or in vivo, to create and/or destroy translation, initiation, 
and/or termination sequences, or to create variations in coding regions 
and/or form new restriction endonuclease sites or destroy preexisting 
ones, to facilitate further in vitro modification. Any technique for 
mutagenesis known in the art can be used, including but not limited to, in 
vitro site-directed mutagenesis (Hutchinson, et al., 1978, J. Biol. Chem. 
253:6551), use of TAB.RTM. linkers (Pharmacia), etc. 
5.6. Uses of Neurite Growth Regulatory Factors 
5.6.1. Diagnostic Uses 
5.6.1.1. CNS Myelin Associated Inhibitory Proteins 
CNS myelin associated inhibitory proteins, analogs, derivatives, and 
subsequences thereof, and anti-inhibitory protein antibodies have uses in 
diagnostics. Such molecules can be used in assays such as immunoassays to 
detect, prognose, diagnose, or monitor various conditions, diseases, and 
disorders affecting neurite growth extension, invasiveness, and 
regeneration. In one embodiment of the invention, these molecules may be 
used for the diagnosis of malignancies. Alternatively, the CNS myelin 
associated inhibitory proteins, analogs, derivatives, and subsequences 
thereof may be used to monitor therapies for diseases and conditions which 
ultimately result in nerve damage; such diseases and conditions include 
but are not limited to CNS trauma, (e.g. spinal cord injuries), 
infarction, infection, malignancy, exposure to toxic agents, nutritional 
deficiency, paraneoplastic syndromes, and degenerative nerve diseases 
(including but not limited to Alzheimer's disease, Parkinson's disease, 
Huntington's Chorea, amyotrophic lateral sclerosis, and progressive 
supra-nuclear palsy). In a specific embodiment, such molecules may be used 
to detect an increase in neurite outgrowth as an indicator of CNS fiber 
regeneration. 
For example, in specific embodiments, the absence of the CNS myelin 
associated inhibitory proteins in a patient sample containing CNS myelin 
can be a diagnostic marker for the presence of a malignancy, including but 
not limited to glioblastoma, neuroblastoma, and melanoma, or a condition 
involving nerve growth, invasiveness, or regeneration in a patient. In a 
particular embodiment, the absence of the inhibitory proteins can be 
detected by means of an immunoassay in which the lack of any binding to 
anti-inhibitory protein antibodies (e.g., IN-1, IN-2) is observed. 
The immunoassays which can be used include but are not limited to 
competitive and non-competitive assay systems using techniques such as 
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" 
immunoassays, precipitation reactions, gel diffusion precipitation 
reactions, immunodiffusion assays, agglutination assays, 
complement-fixation assays, immunoradiometric assays, fluorescent 
immunoassays, protein A immunoassays, immunoelectrophoresis assays, and 
immunohistochemistry on tissue sections, to name but a few. 
In a specific embodiment, ligands which bind to a CNS myelin associated 
inhibitory protein can be used in imaging techniques. For example, small 
peptides (e.g., inhibitory protein receptor fragments) which bind to the 
inhibitory proteins, and which are able to penetrate through the 
blood-brain barrier, when labeled appropriately, can be used for imaging 
techniques such as PET (positron emission tomography) diagnosis or 
scintigraphy detection, under conditions noninvasive to the patient. 
Neurite growth inhibitory factor genes, DNA, cDNA, and RNA, and related 
nucleic acid sequences and subsequences, including complementary 
sequences, can also be used in hybridization assays. The neurite growth 
inhibitory factor nucleic acid sequences, or subsequences thereof 
comprising about at least 15 nucleotides, can be used as hybridization 
probes. Hybridization assays can be used to detect, prognose, diagnose, or 
monitor conditions, disorders, or disease states associated with changes 
in neurite growth inhibitory factor expression as described supra. For 
example, total RNA in myelin, e.g., on biopsy tissue sections, from a 
patient can be assayed for the presence of neurite growth inhibitory 
factor mRNA, where the amount of neurite growth inhibitory factor mRNA is 
indicative of the level of inhibition of neurite outgrowth activity in a 
given patient. 
5.6.1.2. CNS Myelin Associated Inhibitory Protein Receptors 
CNS myelin associated inhibitory protein receptors as well as analogs, 
derivatives, and subsequences thereof, and anti-receptor antibodies have 
uses in diagnostics. These molecules of the invention can be used in 
assays such as immunoassays or binding assays to detect, prognose, 
diagnose, or monitor various conditions, diseases, and disorders affecting 
neurite growth, extension, invasion, and regeneration. For example, it is 
possible that a lower level of expression of these receptors may be 
detected in various disorders associated with enhanced neurite 
invasiveness or regeneration such as those involving nerve damage, 
infarction, degenerative nerve diseases, or malignancies. The CNS myelin 
associated inhibitory protein receptors, analogs, derivatives, and 
subsequences thereof may also be used to monitor therapies for diseases 
and disorders which ultimately result in nerve damage, which include but 
are not limited to CNS trauma (e.g. spinal cord injuries), stroke, 
degenerative nerve diseases, and for malignancies. 
The assays which can be used include but are not limited to those described 
supra in Section 5.6.1.1. 
Neurite growth inhibitory factor receptor genes and related nucleic acid 
sequences and subsequences, including complementary sequences, can also be 
used in hybridization assays, to detect, prognose, diagnose, or monitor 
conditions, disorders, or disease states associated with changes in 
neurite growth inhibitory factor receptor expression. 
5.6.1.3. Metalloproteases and Their Inhibitors 
The metalloproteases of the invention, and their analogs, derivatives, and 
fragments thereof, as well as inhibitors and anti-metalloprotease 
antibodies, may be used for diagnostic purposes. These molecules of the 
invention may be used in assays such as immunoassays or inhibition type 
assays to detect, prognose, diagnose, or monitor various conditions, 
diseases, and disorders affecting neurite growth extension, invasiveness, 
or regeneration. In a specific embodiment, the molecules of the present 
invention can be used to diagnose malignant tumors, in particular, those 
capable of metastasizing to the brain, (e.g., glioblastoma) by detecting 
the presence of or an increase in metalloprotease levels. Alternatively, 
the molecules of the present invention may be used to monitor therapies 
for malignant tumors such as glioblastoma by detecting changes in 
metalloprotease levels. In this latter embodiment, decreases or the 
disappearance of metalloprotease levels should can be indicative of 
treatment efficacy. 
The assays which can be used include but are not limited to those described 
supra in Section 5.6.1.1. 
Metalloprotease genes and related nucleic acid sequences and subsequences, 
including complementary sequences, can also be used in hybridization 
assays, to detect, prognose, diagnose, or monitor conditions, disorders, 
or disease states associated with changes in metalloprotease expression as 
described supra. For example, total RNA in a sample (e.g., glial cells) 
from a patient can be assayed for the presence of metalloprotease mRNA, 
where the presence or amount of metalloprotease mRNA is indicative of a 
malignancy in the patient. In particular, a malignancy that can be 
metastatic to the brain (e.g., glioblastoma) can be detected. 
5.6.2. Therapeutic Uses 
5.6.2.1. CNS Myelin Associated Inhibitory Proteins 
CNS myelin associated inhibitory proteins of the present invention can be 
therapeutically useful in the treatment of patients with malignant tumors 
including, but not limited to melanoma or tumors of nerve tissue (e.g. 
neuroblastoma). In one embodiment, patients with neuroblastoma can be 
treated with the 35 kD and/or 250 kD proteins or analogs, derivatives, or 
subsequences thereof, and the human functional equivalents thereof, which 
are inhibitors of neurite extension. In another embodiment, a patient can 
be therapeutically administered both a CNS myelin-associated inhibitory 
protein and a metalloprotease inhibitor. 
In an alternative embodiment, derivatives, analogs, or subsequences of CNS 
myelin inhibitory proteins which inhibit the native inhibitory protein 
function can be used in regimens where an increase in neurite extension, 
growth, or regeneration is desired, e.g., in patients with nerve damage. 
Patients suffering from traumatic disorders (including but not limited to 
spinal cord injuries, spinal cord lesions, or other CNS pathway lesions), 
surgical nerve lesions, damage secondary to infarction, infection, 
exposure to toxic agents, malignancy, paraneoplastic syndromes, or 
patients with various types of degenerative disorders of the central 
nervous system (Cutler, 1987, In: Scientific American Medicines v. 2, 
Scientific American Inc., NY, pp. 11-1-11-13) can be treated with such 
inhibitory protein antagonists. Examples of such disorders include but are 
not limited to Alzheimer's Disease, Parkinsons' Disease, Huntington's 
Chorea, amyotrophic lateral sclerosis, or progressive supranuclear palsy. 
Such antagonists may be used to promote the regeneration of CNS pathways, 
fiber systems and tracts. Administration of antibodies directed to an 
epitope of CNS myelin associated inhibitory proteins such as the 35 kD 
and/or 250 kD proteins, (or the binding portion thereof, or cells 
secreting such as antibodies) can also be used to inhibit 35 kD and/or 250 
kD protein function in patients. In a particular embodiment of the 
invention, antibodies directed to the 35 kD and/or 250 kD myelin 
associated inhibitory protein may be used to promote the regeneration of 
nerve fibers over long distances following spinal cord damage; in a 
specific example, monoclonal antibody IN-1 may be used. 
Various delivery systems are known and can be used for delivery of CNS 
myelin inhibitory proteins, related molecules, or antibodies thereto, 
e.g., encapsulation in liposomes or semipermeable membranes, expression by 
bacteria, etc. Linkage to ligands such as antibodies can be used to target 
myelin associated protein-related molecules to therapeutically desirable 
sites in vivo. Methods of introduction include but are not limited to 
intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, 
oral, and intranasal routes, and infusion into ventricles or a site of 
tumor removal. Likewise, cells secreting CNS myelin inhibitory protein 
antagonist activity, for example, and not by way of limitation, hybridoma 
cells, encapsulated in a suitable biological membrane may be implanted in 
a patient so as to provide a continuous source of anti-CNS myelin 
inhibiting protein antibodies. 
In addition, any method which results in decreased synthesis of CNS myelin 
inhibitory proteins may be used to diminish their biological function. For 
example, and not by way of limitation, agents toxic to the cells which 
synthesize CNS myelin inhibitory proteins (e.g. oligodendrocytes) may be 
used to decrease the concentration of inhibitory proteins to promote 
regeneration of neurons. 
5.6.2.2. CNS Myelin Associated Inhibitory Protein Receptors 
CNS myelin associated inhibitory protein receptors or fragments thereof, 
and antibodies thereto, can be therapeutically useful in the treatment of 
patients with nerve damage including but not limited to that resulting 
from CNS trauma (e.g., spinal cord injuries), infarction, or degenerative 
disorders of the central nervous system which include but are not limited 
to Alzheimer's disease, Parkinson's disease, Huntington's Chorea, 
amyotrophic lateral sclerosis, or progressive supranuclear palsy. For 
example, in one embodiment, CNS myelin associated inhibitory protein 
receptors, or subsequences or analogs thereof which contain the inhibitory 
protein binding site, can be administered to a patient to "compete out" 
binding of the inhibitory proteins to their natural receptor, and to thus 
promote nerve growth or regeneration in the patient. In an alternative 
embodiment, antibodies to the inhibitory protein receptor (or the binding 
portion thereof or cells secreting antibodies binding to the receptor) can 
be administered to a patient in order to prevent receptor function and 
thus promote nerve growth or regeneration in the patient. Patients in whom 
such a therapy may be desired include but are not limited to those with 
nerve damage, stroke, or degenerative disorders of the central nervous 
system as described supra. 
Various delivery systems are known and can be used for delivery of CNS 
myelin associated inhibitory protein receptors, related molecules, or 
antibodies thereto, e.g., encapsulation in liposomes, expression by 
bacteria, etc. Linkage to ligands such as antibodies can be used to target 
myelin associated protein receptor-related molecules to therapeutically 
desirable sites in vivo. Methods of introduction include but are not 
limited to intradermal, intramuscular, intraperitoneal, intravenous, 
subcutaneous, oral, intranasal routes, and infusion into ventricles or a 
site of tumor removal. 
5.6.2.3. Metalloproteases and Their Inhibitors 
The metalloproteases of the present invention can be therapeutically useful 
in the treatment of various types of nerve damage or degenerative 
disorders of the central nervous system (such as those described supra, 
Section 5.6.2.2) In one embodiment, patients suffering from nerve damage 
resulting from trauma, stroke, or neurodegenerative disorders can be 
treated with the metalloprotease or proteolytically active analogs, 
derivatives, or subsequences thereof which stimulate neurite extension or 
regeneration of CNS fiber. 
In an alternative embodiment, derivatives, analogs, or subsequences of the 
metalloproteases which antagonize or inhibit metalloprotease function, or 
chemical inhibitors of the metalloprotease activity, can be used in 
regimens where an inhibition of invasive migration and spread in the CNS 
is desired. Such inhibitors may include but are not limited to 1,10 
phenanthroline, EDTA, EGTA, cbz-tyr-tyr, cbz-gly-phe-NH.sub.2, 
cbz-phe-phe-NH.sub.2, and cbz-gly-phe-phe-NH.sub.2. 1,10 phenanthroline, 
EDTA, and EGTA may be obtained from commercial vendors (e.g. Sigma 
Chemical Co.). Cbz-tyr-tyr, cbz-gly-phe-NH.sub.2, cbz-phe-phe-NH.sub.2, 
and cbz-gly-phe-phe-NH.sub.2 may also be obtained from commercial vendors 
(e.g. Vega Biotechnologies), or may be chemically synthesized. In specific 
embodiments, patients with various types of malignant tumors, in 
particular, those metastatic to the brain, can be treated with such 
inhibitors. In a preferred embodiment, a patient with a glioblastoma can 
be treated with such inhibitors. In another specific embodiment, 
administration of antibodies directed to an epitope of the metalloprotease 
can also be used to inhibit metalloprotease function in patients. In yet 
another specific embodiment of the invention, metalloprotease inhibitors 
and a CNS myelin associated inhibitory protein can both be administered to 
a patient for the treatment of a malignant tumor, examples of which 
include but are not limited to glioblastoma, neuroblastoma, or a melanoma. 
Various delivery systems are known and can be used for the delivery of 
metalloproteases and related molecules, e.g., encapsulation in liposomes 
or semipermeable membranes, expression by bacteria, etc. Linkage to 
ligands such as antibodies can be used to target molecules to 
therapeutically desirable sites in vivo. Methods of introduction include 
but are not limited to intradermal, intramuscular, intraperitoneal, 
intravenous, subcutaneous, oral, and intranasal routes, and infusion into 
ventricles or a site of tumor removal. 
6. OLIGODENDROCYTES AND CNS MYELIN ARE NONPERMISSIVE SUBSTRATES FOR NEURITE 
GROWTH AND FIBROBLAST SPREADING IN VITRO 
To study the interaction of neurons with central nervous system (CNS) glial 
cells, dissociated sympathetic or sensory ganglion cells or fetal retinal 
cells were plated onto cultures of dissociated optic nerve glial cells of 
young rats. Whereas astrocytes favored neuron adhesion and neurite 
outgrowth, oligodendrocytes differed markedly in their properties as 
neuronal substrates. Immature (O.sub.4.sup.+, A.sub.2 B.sub.5.sup.+, 
GalC.sup.-), oligodendrocytes were frequently contacted by neurons and 
neurites. In contrast, differentiated oligodendrocytes (O.sub.4.sup.+, 
A.sub.2 B.sub.5.sup.-, GalC.sup.+) represented a nonpermissive substrate 
for neuronal adhesion and neurite growth. When neuroblastoma cells or 3T3 
fibroblasts were plated into optic nerve glial cultures, the same 
differences were observed; differentiated oligodendrocytes were 
nonpermissive for cell adhesion, neurite growth, or fibroblast spreading. 
These nonpermissive oligodendrocytes were characterized by a radial, 
highly branched process network, often contained myelin basic protein 
(MBP), and may, therefore, correspond to cells actively involved in the 
production of myelin-like membranes. 
Isolated myelin from adult rat spinal cord was adsorbed to polylysine 
coated culture dishes and tested as substrate for peripheral neurons, 
neuroblastoma cells, or 3T3 cells. Again, cell attachment, neurite 
outgrowth, and fibroblast spreading was strongly impaired. General 
physico-chemical properties of myelin were not responsible for this 
effect, since myelin from rat sciatic nerves favored neuron adhesion and 
neurite growth as well as spreading of 3T3 cells. These results show that 
differentiated oligodendrocytes express non-permissive substrate 
properties, which may be of importance in CNS development or regeneration. 
6.1. Materials and Methods 
6.1.1. Glial Cell Cultures 
Optic nerves were dissected from 7-12 day old or young adult (180-220 g) 
Wistar rats and collected in plating medium (air-buffered enriched 
L.sub.15 with 5% rat serum; Mains and Patterson, 1973, J. Cell Biol. 
59:329-345). The meninges and blood vessels were carefully removed under a 
microscope and the nerves were cut into small pieces. Dissociation of 10 
day old nerves was done for 25 minutes twice in 0.25% trypsin (Sigma) and 
0.02% collagenase (Worthington) (Raff et al., 1979, Brain Res. 
174:283-318) in CMF-PBS (Ca.sup.++ /Mg.sup.++ --free phosphate buffered 
saline) at 37.degree. C. Adult optic nerves were dissociated in 0.1% 
trypsin, 0.1% collagenase for 1 hour at 37.degree. C. followed by 0.5% 
trypsin for 10 minutes. After washing and dissociation by trituration with 
a Pasteur pipet, the cells were plated into the wells of 35 mm tissue 
culture dishes containing four internal wells at a density of 20,000 to 
30,000 cells per well (surface of well: 95 mm.sup.2). For 7-10 day old 
optic nerves the yield of the dissociation was about 10,000 cells per 
nerve. The culture substrate for most of the experiments was polyornithine 
(PORN, Sigma, 0.5 mg/ml in borate buffer, incubated overnight) or 
polylysine (PLYS, Sigma, 50 ng/ml in water); in some experiments, a dried 
collagen film (calf skin collagen, incubation overnight with sterile 
solution), laminin-coated PORN (purified mouse EHS tumor laminin (5 ng/ml, 
incubated for 3 hours on dishes previously coated with PORN), or plain 
tissue culture plastic was used. The culture medium was an enriched 
L.sub.15 medium with 5% rat serum, penicillin (100 U/ml) and streptomycin 
(100 ng/ml) (Mains and Patterson, 1973, J. Cell Biol. 59:329-345). In some 
experiments, 10% fetal calf serum (FCS) was added instead of the rat 
serum. 
Optic nerves of E13 or E17 chicken embryos were dissociated by brief 
trypsin/collagenase treatment and cultured for 2-7 days in L.sub.15 with 
5% FCS on PORN-coated culture dishes. 
6.1.2. Glia--Nerve Cell Co-Cultures 
Three different types of nerve cells were co-cultured with glial cells: 
sympathetic neurons from the superior cervical ganglion of newborn rats, 
sensory neurons from dorsal root ganglia of newborn rats, or cells from 
the retina of E17-E18 embryonic rats. Superior cervical and dorsal root 
ganglia were dissected and dissociated into single cells as described 
(Mains and Patterson, 1973, J. Cell Biol. 59:329-345; Schwab and Thoenen, 
1985, J. Neurosci. 5:2415-2423). Retinas were dissected from the embryos, 
cleaned from adhering blood vessels and incubated in 0.03% trypsin, 0.03% 
DNase for 10 minutes at 37.degree. C., washed by centrifugation in 
serum-containing medium and dissociated by trituration. 
The neurons were added to 2-10 day old glial cultures in the same medium, 
with the addition of NGF (2.5S NGF, 50 or 100 ng/ml) for sensory and 
sympathetic neurons or brain-derived neurotrophic factor for the retinal 
cells (Johnson, J. E. et al., 1986, J. Neurosci. 6:3031-3038). In order to 
suppress the growth of Schwann cells added together with the peripheral 
neurons, pulses of cytosine arabinoside (Ara C, 10.sup.-5 M) were given 
twice for 24 hours on the 2nd and 5th day of co-culture in some 
experiments. The cultures were processed for antibody staining after 1-5 
days of co-culture in the case of retina cells, or after 2 days to 3 weeks 
in the case of peripheral ganglion cells. 
Mouse neuroblastoma cells (line NB-2A) cultured in DMEM/10% FCS were 
detached from the culture flasks by a brief treatment with 0.1% trypsin in 
CMF-Hank's solution terminated by addition of DMEM/FCS. After washing, the 
cells were added to glial cultures (40,000 or 20,000 cells per well) in 
DMEM/FCS with either 2 mM dibutyryl-cyclic AMP or glia-derived neurite 
promoting factor (GdNPF; Guenther et al., 1986, EMBO J. 4:1963-1966). 
Mouse NIH 3T3 cells, treated identically to the neuroblastoma cells, were 
added to 2-3 day old cultures of 7 day old or newborn rat optic nerves at 
a concentration of 20,000 or 40,000 cells per well in DMEM containing 10% 
fetal calf serum or in MEM supplied with insulin (20 ng/ml) and 
transferrin (50 ng/ml). Cultures were returned to the incubator for 2-4 
hours and then fixed with warm 4% formalin in phosphate buffer and double 
stained with the O.sub.1 and O.sub.4 antibodies. 
6.1.3. Immunofluorescence 
The following antibodies as markers for oligodendrocytes, astrocytes, 
neurons or fibroblasts were used: oligodendrocytes: mouse monoclonal 
antibody (mAB) O.sub.4 (Sommer and Schachner, 1981, Dev. Biol. 
83:311-327); mouse mAB O.sub.1 (Sommer and Schachner, 1981, Dev. Biol. 
83:311-327); specific for galactocerebroside (GalC; Singh and Pfeiffer, 
1985, J. Neurochem. 45:1371-1381); goat antiserum against myelin basic 
protein of rabbits (Omlin, et al., 1982, J. Cell Biol. 95:242-248). 
Precursor cells: mouse mAB A.sub.2 B.sub.5 (Sera-Lab, Crawley Down, GB). 
Astrocytes: rabbit antiserum agaist glial fibrillary acid protein (GFAP) 
(Dahl and Bignami, 1976, Brain Res. 116:150-157). Neurons: mouse mAB 
against guinea pig or rabbit neurofilaments (Willard and Simon, 1981, J. 
Cell Biol. 89:198-205). Fibroblasts: mouse mAB Ox7 against Thy-1.1 
(Sera-Lab); goat antiserum against human fibronectin (LETS protein; 
Cappel, N.C.). 
The specific antibodies were visualized by the corresponding anti-mouse, 
anti-rabbit or anti-goat--fluorescein isothiocyanate (FITC) or--rhodamine 
isothiocyanate (RITC) linked secondary antibodies (Cappel, N.C.). Prior to 
staining, the cultures were washed twice with PBS containing 5% sucrose 
and 0.1% bovine serum albumin (BSA). The antibodies O.sub.1, O.sub.4 and 
A.sub.2 B.sub.5 were directed against surface antigens and were therefore 
incubated on the living cultures at room temperature for 30 minutes at a 
dilution of 1:20 in PBS/sucrose/BSA. Antibodies against Thy-1 were diluted 
1:10, anti-fibronectin 1:20. The cultures were then rinsed twice, fixed 
for 10 minutes with 4% formalin in PBS, rinsed again, incubated for 1 hour 
with the labeled secondary antibodies (dilution 1:30 to 1:100), washed and 
mounted in PBS:glycerol (1:1).--For visualization of myelin basic protein 
(MBP) the cultures were briefly fixed in 4% formalin, then treated with 
ethanol/acetic acid and finally incubated with anti-MBP antiserum (1:500 
dilution) for 1 hour at room temperature. Ethanol/acetic acid fixation was 
also used for visualization of neurofilaments. For double labeling 
experiments of A.sub.2 B.sub.5 or O.sub.1 antibodies with the O.sub.4 
antibody, living cultures were first incubated with antibodies A.sub.2 
B.sub.5 or O.sub.1 followed by anti-mouse-FITC, and then with antibody 
O.sub.4 antigen; the sequence was reversed in some experiments. Staining 
the GFAP was done on cultures previously fixed in 95% ethanol/5% acetic 
acid for 30 minutes at 4.degree. C and rehydrated into PBS. In the case of 
O.sub.4 /GFAP double-labeling experiments, staining was first done with 
the O.sub.4 antibody on the living cultures followed by 10 minutes 
fixation in 4% formalin, subsequent ethanol/acetic acid treatment and 
GFAP-staining. For visualization of MBP, the cultures were briefly fixed 
in 4% formalin, then treated with ethanol/acetic acid and finally 
incubated with anti-MBP antiserum (1:500) for one hour at room 
temperature. Ethanol/acetic acid fixation was also used for visualization 
of neurofilaments. 
Double-labeled cultures were evaluated by systematically screening in the 
fluorescence microscope for the presence of one antigen (usually O.sub.4), 
and every labeled cell was examined for the presence of the other antigen, 
e.g. A.sub.2 B.sub.5, O.sub.1, or GFAP. 
6.1.4. Evaluation of Co-Cultures with Nerve Cells, Neuroblastoma Cells, or 
3T3 Cells 
Antibody-labeled cultures were systematically screened in the fluorescence 
microscope and all O.sub.4 -labeled cells were photographed. The same 
fields were photographed under phase contrast illumination. The 
oligodendrocyte surface area occupied by or in contact with neurons, 
neurites, ganglionic Schwann cells, or 3T3 cells was estimated and the 
oligodendrocytes were grouped into 3 categories: cells with &lt;20%, 20%-80%, 
or &gt;80% of the territory covered by neurons, neurites or 3T3 cells. Single 
thin processes, especially of immature cells, were often excluded from the 
evaluation for reason of comparability with the dense process network of 
highly branched oligodendrocytes. In experiments with retinal cells, total 
oligodendrocyte territory and areas overlapped by retinal cells were 
measured with a Hewlett-Packard digitizer. The oligodendrocyte subtypes 
were identified on the corresponding fluorescence micrographs. The 
criteria used for identification were cell morphology and antigenic 
characteristics (O.sub.4 /O.sub.1). A.sub.2 B.sub.5 -staining could not be 
used as a marker for immature cells, since this antigen was rapidly lost 
(without a concomitant change in cell morphology); after coculture with 
neurons. The distinguishing morphological criteria were: shape and size of 
the cell body, number of primary processes, branching pattern of 
processed, and the occurrence of anastomoses and membrane sheets within 
the process network. With these criteria, highly branched oligodendrocytes 
and immature oligodendrocytes could be reproducibly distinguished. Most 
(but not all) of the highly branched cells were positive for the O.sub.1 
antigen; immature cells were consistently negative. 
Quantification of the direction of neuroblastoma process outgrowth with 
respect to highly branched oligodendrocytes was done as illustrated in 
FIG. 5. Highly branched oligodendrocytes were sampled systematically, and 
neighbouring neuroblastoma cells were classified as "adjacent" if the 
distance between the edge of the oligodendrocyte process network and the 
NB-2A cell was less than 2 cell body diameters. Further cells were 
classified as "distant" (FIGS. 4: A-F and 5). A circle with 8 sectors (4 
classes) was overlaid over the center of each neuroblastoma cell, oriented 
towards the nearest oligodendrocyte cell body, and the neuroblastoma 
processes counted in each sector (FIG. 5). 
6.1.5. Preparation of Myelin 
Spinal cords were dissected from 200 g rats, carefully cleaned from 
adhering dorsal and ventral roots, and homogenized (polyton, 30 seconds at 
half maximal speed). Sciatic nerves were dissected, minced and 
homogenized. Myelin fractions were isolated by flotation of low speed 
supernatants on sucrose density gradients (Colman et al., 1982, J. Cell 
Biol. 95:598-608). In some experiments, to remove possible trapped 
contaminants, the crude membrane fraction was washed following hypotonic 
shock. Sedimentation in hypotonic medium was achieved at 10,000.times.g 
for 5 minutes. Membrane fractions in sucrose solutions containing no more 
than 50 mM ionic species were adsorbed for several hours onto the wells of 
PLYS-coated tissue culture dishes (about 0.1 mg of protein per cm.sup.2 of 
tissue culture dish). Unbound membranes were removed by three washes with 
CMF-Hank's solution. Coated dishes were then immediately used in substrate 
testing experiments. In experiments with sympathetic or sensory neurons 
small droplets of central or peripheral myelin were deposited in defined 
patterns over 35 mm culture dishes. 
Sympathetic or sensory neurons cultured as described above were examined 
after 12 hours to 4 days, neuroblastoma cells after 5-24 hours, and 3T3 
cells after 1-4 hours. For quantification, neuroblastoma cells were 
classified as round cells, cells with filopodia or short processes, or 
cells with processes longer than one cell body diameter. 3T3 cells were 
classified as round cells, cells with filopodia or short processes, or 
large flat cells. Three to four micrographs per culture were taken at 
random from 3 cultures for each experimental point. 
6.2. Results 
6.2.1. Cultures of Dissociated Young or Adult Rat Optic Nerves 
GFAP positive astrocytes accounted for about 30% of the cells in 
dissociated 10 day old rat optic nerves. About 50% of the cells were 
positive for the O.sub.4 antigen, a marker for differentiated, 
(GalC-positive) and immature (A.sub.2 B.sub.5 -positive) oligodendrocytes. 
No overlap was seen in the labeling between O.sub.4 and GFAP or O.sub.4 
and Thy-1, confirming the specificity of the O.sub.4 antibody as a marker 
for the oligodendrocyte family (Sommer and Schachner, 1981, Dev. Biol. 
83:311-327). Thy-1-positive fibroblasts with large flat morphologies 
accounted for about 20% of the cells in young rat optic nerves. 
6.2.2. Subtypes of Oligodendrocytes 
In cultures from 7-10 day old rats, about 50% of the O.sub.4 -positive 
cells were A.sub.2 B.sub.5 -labeled cells. Such cells were O.sub.1 
-negative (Table I) and had different morphologies, including cells with 
irregular processes from polygonal cell bodies, flat cells with peripheral 
processes, bipolar cells, or cells decorated with filopodia. On the basis 
of this marker profile (A.sub.2 B.sub.5.sup.+, O.sub.4.sup.+, 
O.sub.1.sup.-) and in agreement with Schnitzer and Schachner (1982, Cell 
Tissue Res. 2245:625-636), we interpret these cells as being precursor and 
immature oligodendrocytes and collectively called them "immature 
oligodendrocytes". This cell group is probably heterogenous, as is also 
suggested by the different morphologies. Filopodia-carrying cells may be 
the most advanced (Table I). 
TABLE I 
______________________________________ 
A: OLIGODENDROCYTE SUBPOPULATIONS (7 DAY 
OPTIC NERVES, 2 DAYS IN CULTURE) DIFFER 
IN THEIR LABELING BY THE ANTIBODY A.sub.2 B.sub.5 
Percentage of Labeled Cells 
A.sub.2 B.sub.5 + /O.sub.4.sup.- 
A.sub.2 B.sub.5 + /O.sub.4.sup.+ 
A.sub.2 B.sub.5.sup.- /O.sub.4.sup.+ 
______________________________________ 
Highly branched 
0 9 .+-. 4 91 .+-. 4 
oligodendrocytes 
Cells with irregular 
or polygonal shapes: 
flat membraneous cells 
37 .+-. 4.sup.a 
51 .+-. 6 12 .+-. 6 
process-bearing cells 
18 .+-. 5 74 .+-. 5 8 .+-. 2 
cells with filopodia 
0 57 .+-. 8 43 .+-. 8 
______________________________________ 
B: OLIGODENDROCYTE SUBPOPULATIONS (7-10 DAY 
OPTIC NERVES, 2 DAYS IN CULTURE) CHARACTERIZED 
BY THE ANTIBODIES O.sub.1 (GalC) and A.sub.2 B.sub.5 * 
Percentage of Labeled Cells 
A.sub.2 B.sub.5 + /O.sub.1.sup.- 
A.sub.2 B.sub.5 + /O.sub.1.sup.+ 
A.sub.2 B.sub.5.sup.- /O.sub.1.sup.+ 
______________________________________ 
Highly branched 
0 7 .+-. 2 93 .+-. 2 
oligodendrocytes 
Cells with irregular 
or polygonal shapes: 
flat membraneous cells 
100 0 0 
process-bearing cells 
84 .+-. 6 14 .+-. 6 1.5 .+-. 1.5 
cells with filopodia 
91 1 (8 .+-. 8).sup.b 
______________________________________ 
*Dissociated 7-10 day old rat optic nerve cells were cultured on PORN for 
2 days and labeled by either first antibody A.sub.2 B.sub.5 (detected by 
antimouse FITC) followed by O.sub.4 or O.sub.1 (detected by 
antimouse-RITC) or vice versa. The proportion of doublelabeled cells was 
calculated from the values obtained for A.sub.2 B.sub.5.sup.+ 
/O.sub.4/1.sup.- and A.sub.2 B.sub.5.sup.+ /O.sub.4/1.sup.+ cells. Values 
represent the means .+-. SEM of 4-6 cultures (120-200 cells/cultures) fro 
2 separate experiments. 
.sup.a) This population of A.sub.2 B.sub.5.sup.+ /O.sub.4/1.sup.- cells 
containing type II astrocytes and precursor cells not expressing any 
oligodendrocyte marker. 
.sup.b) Variable, weak, granular staining 
About 50% of the O.sub.4 -positive cells were A.sub.2 B.sub.5 -negative and 
O.sub.1 -positive after 2 days in culture under our culture conditions. 
Most of these cells showed a typical, highly branched radial process 
network. Due to this characteristic morphology we called these cells 
highly branched oligodendrocytes (Table I). After 2 days in culture, most 
highly branched oligodendrocytes from optic nerves of 10 day old rats were 
stained with an antiserum against myelin basic protein (MBP). We therefore 
interprete these cells as being myelin forming oligodendrocytes. Their 
characteristic process network may be the result of an unstable, partially 
collapsed myelin membrane containing occasional flat membrane areas. The 
total yield of cells from adult nerves was very low. Both, differentiated 
O.sub.1 -positive highly branched oligodendrocytes as well as immature 
A.sub.2 B.sub.5 -positive oligodendrocytes were also present in cultures 
of adult tissue. 
6.2.3. Response of Various Cell Types to Highly Branched Oligodendrocytes 
6.2.3.1. Co-Cultures With Sympathetic or Sensor Neurons 
Dissociated cells from newborn rat superior cervical ganglia or dorsal root 
ganglia were added to glial cells after 2-10 days in culture. Ganglionic 
Schwann cells and fibroblasts were eliminated by pulses of Ara C in some 
of the experiments. NGF (50 or 100 ng/ml) was added to the culture medium, 
leading to a rapid fiber outgrowth and to the formation of dense neurite 
networks within a few days. NGF alone had no effect on the occurrence and 
morphology of oligodendrocytes. Glial cell types were identified by 
antibody staining at the end of the experiments (2 days to 2 weeks of 
co-culture). 
In cultures with a dense neurite plexus, the most striking observation was 
the occurrence of "windows" free of neurites in the center of which cells 
with radial, highly branched processes could be observed (FIGS. 1A-H). 
Antibody staining identified these cells as highly branched 
oligodendrocytes. A quantification of the interaction of oligodendrocytes 
with sympathetic ganglion cells is shown in FIGS. 2A and 2B. Astrocytes 
adjacent to oligodendrocytes were rare in these cultures since the overall 
glial cell density was low; preferential association with astrocytes 
could, therefore, not account for this result. Highly branched 
oligodendrocytes excluded neurons from their territory irrespective of the 
culture substrate used. The same "windows" were formed on plain plastic, 
collagen, PORN- or laminin-coated culture dishes. No difference was seen 
between sympathetic and sensory neurons; both were excluded from the 
territory of highly branched oligodendrocytes. Likewise, Schwann cells, 
when present, did not invade or overgrow the oligodendrocyte process 
networks (FIG. 1B). In contrast, immature oligodendrocytes, characterized 
by their irregular shapes and the absence of O.sub.1 -antigen, did allow 
neurite growth on their processes and cell bodies (FIGS. 1B, 1E, 1F). 
A.sub.2 B.sub.5 could not be used as a marker for immature 
oligodendrocytes in co-cultures with neurons, as this antigen was rapidly 
lost after addition of the neurons. Recent direct observations of the 
encounter of growth cones with oligodendrocytes showed that growth cone 
movement was arrested after filopodial contact is established. Normal 
growth cone activity was seen during contact and crossing of immature 
cells. These observations also exclude the possibility that the "windows" 
were formed secondarily in the neurite plexus. Astrocytes in the same 
cultures were often overgrown by single neurites or neurite bundles (FIGS. 
3A, 3B). This was true for both morphological types, flat and stellate 
cells. 
6.2.3.2. Co-Cultures With Fetal Rat Retinal Cells 
After plating retinal cells at monolayer density on top of 5 day old 
cultures of optic nerve non-neuronal cells, a typical rearrangement of the 
retinal cells could be observed; whereas oligodendrocyte precursor cells 
were often contacted by retina cells, the highly branched oligodendrocytes 
were mostly free of them (FIGS. 1G, 1H, 3C, 3D). Again, astrocytes were 
preferred as a substrate over PORN. 
6.2.3.3. Response of Other Cell Types to Highly Branched Oligodendrocytes 
Neuroblastoma cells (line NB-2A) were plated at high cell density into 
dissociated optic nerve cultures and stimulated for fiber production by 2 
mM dibutyryl-cyclic-AMP or by GdNPF. Seven, 24 or 48 hours later, the 
cultures were fixed and oligodendrocytes were identified by antibodies 
O.sub.4 and O.sub.1. Again, the territories of highly branched 
oligodendrocytes were clearly spared by neuroblastoma cells (FIGS. 4A, 
4B). Processes produced by neuroblastoma cells situated close to 
oligodendrocytes were pointing away from the oligodendrocytes (FIGS. 4A, 
4B; FIG. 5 and Table IA). 
TABLE IA 
______________________________________ 
ORIENTATION OF NEUROBLASTOMA PROCESSES 
WITH REGARD TO HIGHLY BRANCHED OLIGODENDROCYTES 
% of Processes in Each Sector 
Adjacent Neuro- 
Distant Neuro- 
Sector.dagger. 
blastoma Cells 
blastoma Cells 
______________________________________ 
1 7 .+-. 1.4 25 .+-. 2.4*** 
2 34 .+-. 1.2 26 .+-. 1.2*** 
3 33 .+-. 2.7 25 .+-. 2.3* 
4 26 .+-. 2.3 24 .+-. 2.7 
______________________________________ 
.dagger.Shown in FIG. 5 
*p &lt; 0.0.05 
***p &lt; 0.001 
Primary culture fibroblasts and astrocytes in the optic nerve preparations 
as well as mouse 3T3 cells showed a drastic "avoidance behavior" towards 
highly branched oligodendrocytes. 3T3 cells plated at high cell density 
into optic nerve glial cultures attached and flattened out between 30 
minutes and 3 hours on the PORN substrate. In these forming monolayers, 
characteristic "windows" appeared corresponding to the territories of 
highly branched oligodendrocytes (FIGS. 4C, 4D). At the sites of contact, 
3T3 cells formed a crescent-shaped bulge of cytoplasm. Lamellipodia were 
absent in this region. Significantly, fibroblasts that landed directly on 
highly branched oligodendrocytes completely failed to spread. As for 
neurons, immature oligodendrocytes were not visibly avoided by 3T3 cells 
(FIGS. 6A-B). 
6.2.4. Absence of Species Specificity 
Neither the specific morphology nor the unfavorable substrate property of 
oligodendrocytes were species specific. Dissociated non-neuronal cells 
from E13 and E17 chick optic nerve contained besides O.sub.4 
-positive/A.sub.2 B.sub.5 -negative/O.sub.1 -positive highly branched 
oligodendrocytes. 3T3 cells plated on top of chicken non-neuronal cells 
formed the characteristic "windows" around these chick oligodendrocytes. 
6.2.5. Myelin as a Substrate 
The properties of myelin as a substrate for neurons or fibroblasts were 
also tested, since myelin consists of spirally wrapped oligodendrocyte 
membranes. Crude myelin fractions from adult rat spinal cord or sciatic 
nerve were prepared by flotation on a sucrose gradient. The myelin was 
adsorbed to PLYS-coated tissue culture dishes and tested for its substrate 
properties for superior cervical ganglion cells, dorsal root ganglion 
cells, neuroblastoma cells and 3T3 cells. All four cell types attached 
poorly to CNS myelin and showed marked difficulties in their process 
outgrowth. Sympathetic and sensory neurons on CNS myelin remained round or 
produced short, abortive fibers in spite of the presence of NGF (50 ng/ml 
or 100 ng/ml) (FIGS. 7A, 17C). In contrast, long fibers were produced on 
islets of sciatic nerve myelin in the same culture dishes (FIGS. 7B, 17D). 
Small CNS myelin islets on PLYS appeared as "windows" outlined by excluded 
neurites, whereas PNS myelin-PLYS boundaries were apparently not detected 
by growing neurites. 
Process outgrowth from neuroblastoma cells (line NB-2A) in the presence of 
dibutyryl-cyclic AMP was significantly reduced by CNS myelin (FIG. 8A). 
Spreading of 3T3 fibroblasts was strongly inhibited by CNS myelin (FIG. 
8B). 3T3 cells remained round or produced spindle-shaped or polygonal 
morphologies with a minimal cell substrate interaction. In contrast, large 
flat membranes were produced within 20-30 minutes on polylysine and, with 
a somewhat slower time-course, also on myelin from the peripheral nervous 
system (FIG. 8B). Nonpermissiveness was associated, at least in large 
part, with myelin membranes, since sedimentation at 10,000.times.g for 5 
minutes under hypotonic conditions (see Section 6.1.5., supra) was 
sufficient to pellet most nonpermissive membranes. Under these conditions, 
most surface membrane components floating to densities smaller than the 
one of 0.85M sucrose, would not be expected to sediment. 
CNS myelin nonpermissiveness is not due to astrocyte membranes, since a 
cell membrane preparation from CNS tissue containing minimal amounts of 
white matter (superficial cortical layers) was a permissive substrate for 
fibroblast spreading. 
These experiments show that, in parallel to the effects of living, highly 
branched oligodendrocytes, myelin from the CNS is also a strongly 
nonpermissive substrate for primary culture neurons, neuroblastoma cells, 
and 3T3 fibroblasts. Myelin from the peripheral nervous system does not 
show a comparable nonpermissive substrate effect. 
6.3. Discussion 
In the present study, we observed that myelin forming oligodendrocytes and 
isolated CNS myelin exert a nonpermissive substrate effect on outgrowing 
neurites of sympathetic and sensory neurons and neuroblastoma cells, as 
well as for the attachment of retinal cells and the spreading of 
fibroblasts. 
Several classes of cells were present in short-term cultures of dissociated 
rat optic nerves: oligodendrocytes, astrocytes (GFAP-positive) fibroblasts 
(Thy-1, fibronectin-positive) and several types of precursor cells. Within 
the oligodendrocyte family (O.sub.4 -positive; Sommer and Schachner, 1981, 
Dev. Biol. 83:311-327), one main subtype of cells was characterized by the 
absence of the O.sub.1 antigen (GalC) and of MBP, two components highly 
characteristic of myelin (Mirsky, et al., 1980, J. Cell Biol. 84:483-494), 
and the presence of binding sites for the antibody A.sub.2 B.sub.5. 
A.sub.2 B.sub.5 was shown to be a marker for oligodendrocyte/type II 
astrocyte precursors, type II astrocytes, and neurons (Schnitzer and 
Schachner, 1982, Cell Tissue Res. 224:625-636; Abney, E. R. et al., 1981, 
Dev. Biol. 100:166-171; Raff, et al., 1983, Nature 303:390-396). 
Therefore, we considered this cell class to represent immature 
oligodendrocytes, probably including precursors such as those described by 
Dubois-Dalcq (1986, Soc. Neurosci. Abstr. 12:767) and Sommer and Noble 
(1986, Soc. Neurosci. Abstr. 12:1585). The presence of O.sub.4 
distinguishes these cells from the O2A precursors (Raff, M. C. et al., 
1983, Nature 303:390-396). These immature cells showed irregular and 
variable morphologies with bipolar shapes or polygonal cell bodies and 
irregular processes, often decorated with filopodia. The cell class is 
probably heterogenous; cell division could be observed. The second main 
oligodendrocyte subclass consisted of A.sub.2 B.sub.5 -negative, O.sub.1 
-positive cells, possessing a radial, highly branched and anastomosing 
process network. Most of these highly branched oligodendrocytes in 2 day 
old cultures of 10 day old rat optic nerves were positive for MBP under 
our culture conditions. We thus interpret this frequent cell type as 
representing oligodendrocytes actively involved in the synthesis of myelin 
membranes which are deposited flat on the culture substrate in the absence 
of axons. These membranes are unstable and collapse to form the 
characteristic, anastomosing process network. This cell type has been 
described as "hairy eyeball cell" (Sommer and Schachner, 1981, Dev. Biol. 
83: 311-327), and formation of whorls of compact myelin by such cells has 
been observed after prolonged times in culture (Rome et al., 1986, J. 
Neurosci. Res. 15:49-65; Yim et al., 1986, J. Biol. Chem. 
261:11808-11815). 
Both immature and myelin forming oligodendrocytes were seen in cultures of 
7 to 10 day old or adult rat optic nerves, and also in cultures of 1 day 
rat optic nerves, newborn rat spinal cord or adult rat corpus callosum, as 
well as in cultures of spinal cord and optic nerves of E13 or E17 chicken 
embryos. Immature cells clearly were predominant in dissociates from 
younger stages, but the large drop in cell yield upon dissociation with 
increasing age precluded any quantitative population analysis. However, 
immature oligodendrocytes could also be obtained consistently from adult 
rat white matter tissues, confirming earlier observations by 
French-Constant and Raff (1986, Nature 319:499-502). 
The addition of neurons to established glial cultures showed dramatic 
differences in substrate properties for neuronal attachment and fiber 
outgrowth among the various types of non-neuronal cells. Astrocytes, 
particularly the flat reactive protoplasmic astrocytes, were adhesive and 
favorable for neuronal attachment and outgrowth, in agreement with earlier 
observations (Foucaud et al., 1982, Cell Res. 137:285-294; Hatten, et al., 
1984, J. Cell Biol. 98:193-204; Noble, et al., 1984, J. Neurosci. 
4:1982-1903; Fallon, 1985, J. Cell Biol. 100:198-207). Immature 
oligodendrocytes also were frequently contacted by neurites or nerve cell 
bodies. This behavior could be of high physiological relevance. During 
development, oligodendrocyte precursors migrate into the already formed 
axonal bundles and extend processes to contact a certain number of axons. 
These processes then start to enwrap and spiral around the axons, thus 
forming the structure called myelin (Wood and Bunge, 1984, W. T. Norton, 
ed., 1-46). 
In sharp contrast to astrocytes and oligodendrocyte precursors, we found 
that myelin forming oligodendrocytes display strongly nonpermissive 
substrate properties for neuronal attachment and fiber outgrowth as well 
as for fibroblast attachment and spreading. This effect was strong and 
pronounced even on laminin-coated culture dishes, which otherwise 
represent an excellent substrate for neurite growth (Manthorpe, et al., 
1983, J. Cell Biol. 97:1882-1980; Rogers, et al., 1983, Dev. Biol. 
98:212-220). This effect was not overcome by high doses of NGF in cultures 
of sympathetic and sensory neurons, or GdNPF or dibutyrylcyclic-AMP in 
cultures of neuroblastoma cells. A similar or identical nonpermissive 
substrate property was associated with rat CNS myelin but not with myelin 
from peripheral nerves. The effect was strictly contact-dependent, since 
nerve cells or fibroblasts grew well and were free to move in the 
immediate surrounding of these oligodendrocytes or of CNS myelin islets. 
Mouse 3T3 cells were also inhibited by chicken oligodendrocytes, showing 
that this effect is not species-specific. 
In the rat optic nerve, the peak number of axons is reached at embryonic 
day 20, followed by a dramatic loss of axons (Crespo, et al., 1985, Dev. 
Brain Res. 19:129-134). Oligodendrocyte precursors appear from E17 onward 
(Raff, et al., 1985, Cell 42: 61-69) and express Ga1C around birth 
(Miller, et al., 1985, Dev. Biol. 111: 35-41). The first myelin detectable 
by electron microscopy appears at postnatal day 6 (Hildebrand and Waxman, 
1984, J. Comp. Neurol. 224: 25-37). This clear-cut temporal dissociation 
between axonal growth and myelin formation is also present in chicken 
optic nerves (Rager, 1980, Cell Biol. 63: 1-92) and, although less well 
studied, in many white matter tracts of the CNS (Matthews and Duncan, 
1971, J. Comp. Neurol. 142: 1-22; Looney and Elberger, 1986, J. Comp. 
Neurol. 248: 336-347). During normal development, growing axons therefore 
probably never encounter myelin or myelinating oligodendrocytes within 
their fascicles, but rather interact with precursors and immature 
oligodendrocytes. The extremely slow time-course observed for in vitro 
myelination (Wood, et al., 1980, Brain Res. 196: 247-252; Wood and 
Williams, 1984, Dev. Brain Res. 12: 225-241) could be consistent with a 
situation where undifferentiated oligodendrocytes first interact with 
axons and are then induced to differentiate and to form myelin. 
In contrast to development, during CNS regeneration, axonal growth cones or 
sprouts do encounter mature oligodendrocytes and myelin. Substrate 
properties of CNS tissue, in particular the absence of potent neurite 
promoting substrates like laminin in the differentiated CNS of higher 
vertebrates, are important aspects in the context of CNS regeneration 
(Liesi, 1985, EMBO J. 4: 2505-2511; Carbonetto, et al., 1987, J. Neurosci. 
7: 610-620). However, since myelin and oligodendrocytes persist for a long 
time in denervated CNS tracts (Fulcrand and Privat, 1977, J. Comp. Neur. 
176: 189-224; Bignami, et al., 1981, J. Neuropath, Exp. Neurol. 40: 
537-550), the absence of any fiber regeneration in white matter areas in 
contrast to peripheral nerves and PNS/CNS transplants could be related to 
these nonpermissive substrate factors. 
Under normal conditions, blocking certain territories for later growing 
axonal populations during development, antagonism between favorable and 
nonpermissive substrate molecules during development of CNS projection 
patterns, or the spatial limitation of sprouting in the differentiated CNS 
are possible functions for this oligodendrocyte associated nonpermissive 
substrate property. 
7. TWO MEMBRANE PROTEIN FRACTIONS FROM CENTRAL NERVOUS SYSTEM MYELIN WITH 
INHIBITORY PROPERTIES FOR NEURITE GROWTH AND FIBROBLAST SPREADING 
We have searched for surface components in CNS white matter, which would 
prevent neurite growth. CNS, but not PNS, myelin fractions from rat and 
chick were highly nonpermissive substrates in vitro. We have used an in 
vitro spreading assay with 3T3 cells to quantify substrate qualities of 
membrane fractions and of isolated membrane proteins reconstituted in 
artificial lipid vesicles. CNS myelin nonpermissiveness was abolished by 
treatment with proteases and was not associated with myelin lipid. 
Nonpermissive proteins were found to be membrane bound and yielded highly 
nonpermissive substrates upon reconstitution into liposomes. Size 
fractionation of myelin protein by SDS-PAGE revealed two highly 
nonpermissive minor protein fractions of molecular weight, 35 kD and 250 
kD. Removal of 35 kD and of 250 kD protein fractions yielded a CNS myelin 
protein fraction with permissive substrate properties. Supplementation of 
permissive membrane protein fractions (PNS, liver) with low amounts of 35 
or of 250 kD CNS myelin protein was sufficient to generate highly 
nonpermissive substrates. Inhibitory 35 and 250 kD proteins were found to 
be enriched in CNS white matter and were found in optic nerve cell 
cultures which contained highly nonpermissive, differentiated 
oligodendrocytes. 
The data presented herein (Caroni and Schwab, 1988, J. Cell Biol. 106: 
1281-1288) demonstrate the existence of 35 kD and 250 kD myelin 
membrane-bound proteins with potent nonpermissive substrate properties. 
Their distribution and properties suggest that these proteins might play a 
crucial inhibitory role during development and regeneration in CNS white 
matter. 
7.1. Materials and Methods 
7.1.1. Cell Culture 
Mouse NIH 3T3 cells were cultured and assayed for spreading behavior in 
DMEM containing 10% FCS. In control experiments, use of defined serum-free 
medium did not alter responses of 3T3 cells to tested substrates. Mouse 
neuroblastoma cells (line NB-2A) were cultured in DMEM with 10% FCS in the 
presence of either 1 mM dibutyryl-cAMP or of glia-derived neurite 
promoting factor (GdNPF). Superior cervical and dorsal root ganglia from 
newborn rats were dissected and dissociated into single cells as described 
(Mains and Patterson, 1973, J. Cell Biol. 59: 329-345; Schwab and Thoenen, 
1985, J. Neurosci. 5: 2415-2423). Neurons were cultured in an enriched L15 
medium with 5% rat serum (Mains and Patterson, 1973, J. Cell Biol. 59: 
329-345) and with 100 ng/ml of 2.5S nerve growth factor. Overgrowth by 
contaminating dividing cells was prevented by inclusion of cytosine 
arabinoside (10.sup.-5 M) in the culture medium. 
7.1.2. Sources of Tested Substrates 
Myelin fractions were all prepared by the same procedure, involving tissue 
homogenization in isotonic sucrose buffer with a polytron (model PCU-2; 
Kinematica, Luzern, Switzerland) homogenizer (setting 4, two times 30 s, 
on ice) and flotation of a low speed supernatant onto a discontinuous 
sucrose gradient (myelin collected at 0.25M sucrose top layer) (Colman, et 
al., 1982, J. Cell Biol. 95: 598-608). All isolation media contained 
trasylol (aprotinin; Sigma Chemical Co., St. Louis, Mo.; 100 U/ml), 5 mM 
iodoacetamide, and 5 mM EDTA to reduce protease digestion (this reagent 
mixture is designated below as protease inhibitors). Finally, myelin 
fractions were washed hypotonically in 30 mM Hepes (pH 7.4) (medium A) 
plus protease inhibitors and frozen in aliquots at -80.degree. C. CNS 
myelin fractions were prepared from spinal cords carefully stripped of 
ventral and dorsal roots, or from rat optic nerves. The following sources 
were used: rat, spinal cord, 3 month old Lewis rats, male; chick, spinal 
cord, P21; trout, spinal cord, 2 year; frog, spinal cord, 6 months. PNS 
myelin fractions were prepared from rat sciatic nerves (3 month male). Rat 
liver cell membranes were prepared by standard procedures, involving mild 
isotonic homogenization and collection of membranes at the density of 
0.25M sucrose (discontinuous sucrose gradient). Rat CNS tissue enriched in 
gray matter was obtained from superficial neocortex layers, whereas white 
matter-enriched tissue consisted of the corpus callosum. 
7.1.3. Substrate-Assaying Procedure 
Substrates to be tested, in 40-70 mOsmol solutions, were dried onto 
polylysine (PLYS)-coated tissue culture dishes. Unbound membranes and 
solutes were removed by three washes with Ca.sup.2+ Mg.sup.2+ -free Hank's 
solution. Coated dishes were then immediately Used in substrate-testing 
assays. For most experiments, substrates were dried onto the wells of 
dishes (35-mm dishes with four internal wells; Greiner, Nurtingen, Federal 
Republic of Germany). 3T3 cells were detached from -30% confluent cultures 
by brief trypsin (0.2%) treatment in 37.degree. C. PBS plus EDTA. 
Trypsinization was stopped by 10-fold excess of serum-containing DMEM; 
cells were collected and resuspended in DMEM-10% FCS at appropriate 
concentrations. 30,000 cells/cm.sup.2 were added to precoated culture 
dishes, and experiments were scored after 1 hour in culture. In some 
cases, due to occasional slower spreading behavior of 3T3 cell 
populations, scoring had to be delayed up to 2 hours in culture. After 
periods of more than .about.5 hours, inhibitory properties of myelin 
fractions in the absence of serum were less pronounced than observed in 
the presence of 10% FCS, possibly due to the presence of substrate 
digesting proteases. If substantial spreading on PLYS-coated dishes was 
not obtained within 2 hours in culture, tests were discarded and repeated 
with a fresh batch of cells. Quantitative evaluation of spreading was 
performed with a surface integration program on at least 30 cells per 
experimental point. Only spread cells were considered and a zero-spreading 
value (strongly refractory and round cells) was subtracted. Each 
experiment was repeated at least five times. Experiments were found to be 
subject to only small quantitative variations, and values from 
representative experiments are given. Spreading degrees are given as means 
.+-. standard error of the mean. When recoveries of inhibitory activity 
were estimated, serial dilutions of liposomes (in medium A) were assayed 
for nonpermissiveness. In some cases, in order to detect differences among 
strong inhibitory substrates, 3T3-spreading times were extended to up to 5 
hours. Recovery values are based on internal calibration with a CNS myelin 
liposome standard, and are to be considered as first approximations. When 
neurite extension was evaluated, neuroblastoma cells or superior cervical 
ganglia neurons were seeded at .about.25,000 cells/cm.sup.2 and 
experiments were scored after 24 hours in culture. 
7.1.4. Substrate Processing 
Protease sensitivity of inhibitory fractions was determined by digesting 
washed, protease inhibitor-depleted membranes with trypsin. Membrane 
fractions (concentrations of maximally 1 mg of protein per ml) were 
exposed for 10 minutes at room temperature to 0.1% trypsin. Digestion was 
interrupted by the addition of 0.2% trypsin inhibitor (Sigma Chemical Co.) 
and membranes were either washed in medium A or separated from protease by 
Sephadex G-50 chromatography (liposomes). Under these conditions, trypsin 
was retarded by the column, whereas liposomes were recovered in the 
excluded volume. Digested, washed membranes were finally adsorbed to 
culture dishes, and their substrate properties were analyzed as described 
supra. In some experiments with myelin fractions, pronase (Sigma Chemical 
Co.) or elastase (Sigma Chemical Co.) were used. In those instances, 
protease was removed by three washes of the myelin in 30 mM Hepes, pH 7.4. 
Extraction of peripheral membrane proteins from CNS myelin was performed by 
resuspending membranes in either 4M guanidinium chloride (Merck & Co., 
Inc. Rahway, N.J.)/30 mM Hepes or in 500 mM unbuffered Tris base (Sigma 
Chemical Co.). Protease inhibitors were routinely included in the 
extraction buffers. After incubation for 30 minutes at room temperature, 
myelin was sedimented, washed in medium A, and assayed for its substrate 
properties. 
Ethanol/ether (2:3 vol ratios) extraction of myelin was performed by a 
standard procedure (see, for example, Everly, J. L. et al., 1973, J. 
Neurochem. 21: 329-334). Solvent-insoluble fraction was reconstituted into 
lipid vesicles (see Section 7.1.5,) infra). The lipid-containing soluble 
fraction was dried and reconstituted by the cholate method (see Section 
7.1.5, infra). 
7.1.5. Liposomes 
Liposomes were prepared in medium A by the cholate method (Brunner, et al., 
1978, J. Biol. Chem. 253: 7538-7546). Protein was solubilized in 2% SDS 
(in medium A plus protease inhibitors); insoluble protein was sedimented 
and discarded. Solubilized protein was precipitated with a 30-fold excess 
of acetone. To obtain reproducible yields, acetone precipitation was 
allowed to proceed for 15 hours at 4.degree. C. Protein extracts from 
tissues were prepared by homogenization of minced tissue with a 
glass-teflon potter in 2% SDS-containing, protease 
inhibitors-supplemented, medium A. Solubilized protein was then 
precipitated with ice-cold acetone as described above. Extracts from 
cultured cells were prepared by, first, detaching the cells with a rubber 
policeman in the presence of PBS plus EDTA plus protease inhibitors, and 
by then homogenizing suspended cells with a glass-teflon potter. Upon 
low-speed pelleting of nuclear material, 2% SDS was added to supernatants 
and solubilized protein was precipitated with ice-cold acetone. In all 
cases, acetone-precipitated protein was sedimented (10,000.times. g, 15 
minutes) and resuspended at 1 mg/ml in medium A with 2.5% cholate. 
Phospholipids (phosphatidylcholine/phosphatidylserine, 10:1) dissolved in 
medium A with 2.5% cholate were then added (.about.5-10:1 ratio of added 
phospholipid to protein) and liposomes were formed on a Sephadex G-50 
column. When gel-extracted protein was reconstituted, precipitated protein 
was resuspended at .about.50 .mu.g/ml and phospholipid to protein ratios 
were up to 100:1. 
A number of control experiments were performed. Thus, acetone precipitated 
myelin or gel-extracted protein did not prevent 3T3 spreading when 
resuspended in medium A or in medium A with 2.5% cholate and adsorbed 
directly onto PLYS-coated dishes. Also, running of cholate-solubilized 
protein on the Sephadex G-50 column, in the absence of phospholipids did 
not yield nonpermissive fractions. Experiments with .sup.125 I-labeled CNS 
myelin protein showed that .about.30% of applied label was recovered in 
the liposome fraction from the G-50 column. In some experiments, lipid 
vesicles were formed in the presence of trace amounts of .sup.3 H! 
cholesterol and were then dried onto the wells of tissue culture dishes 
(Greiner). Total culture dish associated membrane amounts (.sup.3 H! 
cholesterol) were found to vary independently of tested protein, 
indicating that differences in liposome binding to culture dishes cannot 
be responsible for the observed differences in substrate properties. 
7.1.6. Gel-Extracted Protein Fractions as Substrate 
Protein was run on 3-15% gradient gels under reducing conditions. For this 
purpose, samples were preincubated for 30 minutes at room temperature in 
sample buffer containing 2% SDS and .beta.-mercaptoethanol. Thin lanes 
were cut and stained with Coomassie Brilliant Blue or with the silver 
method. Protein bands to be analyzed were carefully aligned with the 
unstained gel parts to be extracted. Gel regions from the unstained gel 
part were cut, and minced gel was extracted for 1 hour with 0.5% SDS. In 
most experiments, 50 .mu.g/ml of insulin (Sigma Chemical Co.) were 
included in order to reduce losses due to adsorption of protein present in 
low concentration. Insulin was selected for its purity and for its small 
size, resulting in efficient separation from the liposome fraction. In 
control experiments, no substrate differences could be detected when 50 
.mu.g/ml of insulin were added to various reconstitution mixtures, 
including protein-free liposomes. Gel-extracted protein was precipitated 
with a 10-fold excess of ice-cold acetone (15 hours), and sedimented 
protein was resuspended in cholate buffer. Protein was stored frozen in 
cholate buffer and reconstitution mixtures were prepared from these 
protein stocks. Reconstitution and test of substrate properties were 
performed as described above. 
The amount of protein present was determined by the filter binding method 
(Schaffner and Weissman, 1973, Anal. Biochem. 56: 502-514) with BSA (Sigma 
Chemical Co.) as a standard. 
7.2. Results 
7.2.1. Nonpermissive Substrate Effect is Found in CNS Myeline of Higher 
Vertebrates (Chick, Rat), but Not of Lower Vertebrates (Trout, Frog) 
Rat CNS myelin was found to be a nonpermissive substrate for neurite 
outgrowth from rat superior cervical ganglion neurons and for spreading 
and migration of 3T3 fibroblasts (see also section 6, supra). Analogous 
results were obtained when myelin was prepared from rat optic nerve or 
from rat brain. Neuron type apparently did not influence substrate 
response as similar results were obtained with dorsal root ganglion 
neurons. Likewise, rat CNS myelin nonpermissiveness was observed for 
dibutyryl-AMP-induced or GdNPF-induced outgrowth from neuroblastoma cells. 
Thus, nonpermissiveness of rat CNS myelin is apparently general with 
regard to neuron type and induction of neurite outgrowth. 
Lack of regenerative fiber growth is found in the CNS of higher vertebrates 
but not in those of fishes and to a limited extent in those of amphibia 
(Bohn, et al., 1982, Am. J. Anat. 165: 307-419; Stensas, 1983, In Spinal 
Cord Reconstruction, C. C. Kao, R. P. Bunge, and P. J. Reier, eds., Raven 
Press, New York 121-149; Hopkins, et al., 1985, J. Neurosci. 5: 3030-3038; 
Liuzzi and Lasek, 1986, J. Comp. Neurol. 247: 111-122). We prepared spinal 
cord myelin fractions from trout, frog, and chick in order to determine 
potential differences in substrate properties. Trout (FIG. 9) and frog 
(FIG. 9) CNS myelin fractions were found to have substrate properties 
similar to those of rat PNS myelin, whereas CNS myelin from the chick 
(spinal cord, postnatal 21) was a nonpermissive substrate, although 
slightly less so than its rat counterpart. 
7.2.2. Membrane-Bound Protein Fraction of Rat CNS Myelin is Responsible for 
its Nonpermissive Substrate Properties 
Rat CNS myelin was processed by standard procedures in order to determine 
the nature of the component(s) responsible for its nonpermissive substrate 
properties. Fractions were tested for reduction of 3T3 fibroblast 
spreading. Data are shown in Table II. 
TABLE II 
______________________________________ 
NONPERMISSIVENESS OF CNS MYELIN 
IS DUE TO MEMBRANE-BOUND PROTEIN* 
3T3 spreading 
(.mu.m.sup.2) 
______________________________________ 
Substrate 
Tissue culture plastic 
1,646 .+-. 309 
CNS myelin 
Untreated 211 .+-. 30 
Trypsin-treated 1,344 .+-. 181 
Liposomes 
Ethanol/ether-soluble myelin fraction 
1,253 .+-. 159 
Ethanol/ether-insoluble myelin fraction 
226 .+-. 45 
Artificial lipid vesicles, no additions 
1,328 .+-. 136 
______________________________________ 
*Spreading extent of 3T3 cells was estimated after 1 hour in culture. 
Protein amounts to be adsorbed to wells of dishes (Grenier) were 20 .mu.g 
of CNS myelin protein per cm.sup.2. In the solvent extraction experiments 
100 .mu.g of CNS myelin protein were extracted and onefifth of resulting 
liposomecontaining volume was dried onto wells. These myelin quantities 
represent about 10 times saturation levels with respect to observed 
nonpermissiveness. 
Brief treatment of the myelin with trypsin abolished nonpermissiveness. 
Similar results were obtained with elastase or with pronase treatment. 
Extraction of the myelin under conditions that solubilize peripheral 
membrane proteins (4M guanidinium chloride or pH 10.5) failed to 
dissociate nonpermissiveness from low speed myelin membrane pellets. Lipid 
extraction with ethanol/ether yielded a permissive lipid fraction and a 
nonpermissive protein fraction (Table II). The latter required detergent 
to be solubilized and had to be incorporated into lipid vesicles in order 
to permit detection of nonpermissive substrate property. In control 
experiments, phosphatidylcholine/phosphatidylserine liposomes were a 
slightly less favorable substrate than tissue culture plastic (Table II). 
When CNS myelin protein-containing liposomes were subjected to trypsin 
treatment, their nonpermissive substrate properties were abolished (Table 
III). Thus, a membrane-bound protein fraction from rat CNS myelin is a 
nonpermissive substrate for 3T3 fibroblast spreading. That fraction can 
apparently be reconstituted in active form into artificial lipid vesicles. 
In control experiments, protein from membrane fractions with permissive 
substrate properties yielded, upon reconstitution, liposomes that were 
permissive for 3T3 spreading (Table III). 
TABLE III 
______________________________________ 
NONPERMISSIVE SUBSTRATE PROPERTY OF CNS 
MYELIN IS PRESERVED UPON RECONSTITUTION INTO 
ARTIFICIAL LIPID VESICLES 
Dish-Adsorbed 
Reconstituted 3T3 Spreading 
Lipids (cpm 
Protein Fraction 
(.mu.m.sup.2) 
.sup.3 H!cholesterol) 
______________________________________ 
No protein 1,638 .+-. 91 
521 .+-. 65 
CNS myelin 136 .+-. 30 
650 .+-. 58 
CNS myelin; resulting 
1,397 .+-. 152 
630 .+-. 32 
liposomes trypsinized 
PNS myelin 1,570 .+-. 136 
620 .+-. 41 
Liver membranes 
1,445 .+-. 121 
750 .+-. 47 
______________________________________ 
*Tested protein fractions (100 .mu.g) were reconstituted and onefifth of 
resulting liposomecontaining volume (60 .mu.l) was adsorbed to wells. The 
adsorbed volume contained 20,000 cpm .sup.3 H!cholesterol. Dishadsorbed 
counts were determined upon SDS solubilization of adsorbed liposomes. For 
these experiments, liposomes were removed prior to fibroblast addition. 
Trypsinisation of CNS myelin liposomes and separation of inhibitorblocked 
trypsin from vesicles was performed as described in Section 7.1.4, supra. 
7.2.3. Identification of 35 kD and 250 kD Minor Proteins from Myelin as 
Nonpermissive Substrates for Fibroblast Spreading and Neurite Outgrowth 
As myelin nonpermissiveness partially survived denaturing procedures, 
attempts were made to identify responsible components following separation 
by SDS-PAGE. In preliminary experiments, it was found that solubilization 
of myelin proteins in SDS-PAGE sample buffer followed by reconstitution of 
acetone-precipitated protein yielded a fraction possessing .about.30% of 
starting nonpermissiveness. Apparent activity recoveries were estimated by 
assaying serial dilutions of reconstituted protein with the 3T3 fibroblast 
spreading assay. As a comparison, solubilization in 2% NP-40.TM., 0.5% 
Na-deoxycholate yielded apparent activity recoveries of .about.80%. When 
CNS myelin protein was run on SDS-PAGE and the entire gel was then 
extracted with 0.5% SDS, recoveries of nonpermissive substrate activity 
were .about.20%. Activity could be recovered in approximately equal 
amounts .about.10% of applied activity) from gel regions corresponding to 
the migration distance of 35 kD and of 250 kD proteins, respectively (FIG. 
10). The inhibitory proteins were highly effective, as just 10 ng of 250 
kD protein per cm.sup.2 of culture dish was required to obtain 
half-maximal inhibition. Neither the 250 kD nor the 35 kD region contained 
major myelin protein bands (each region contained .about.3% of total 
silver stained myelin protein). These gel regions apparently contained 
more than one protein species. Reconstitution of pooled gel regions 
depleted of 35 kD and of 250 kD proteins yielded permissive liposomes 
(FIGS. 11A-C). Thus, 35 kD and 250 kD proteins account for most of the 
nonpermissive substrate activity of gel-extracted CNS myelin protein. 
Similarly to unfractionated myelin, 35 kD and 250 kD proteins were 
nonpermissive substrates for fibroblast spreading and for neurite 
extension (FIGS. 11A-C). In control expriments, sciatic nerve protein or a 
liver homogenate did not generate 250 kD nor 35 kD nonpermissive protein 
fractions (FIG. 12). It seems, therefore, reasonable to conclude that the 
protein fractions identified above are responsible for the marked 
nonpermissive substrate properties of rat CNS myelin in vitro. 
We next asked whether addition of these proteins to fractions with neutral 
substrate properties is sufficient to generate a nonpermissive substrate. 
As shown in FIG. 12, liver protein and sciatic nerve protein could yield 
nonpermissive substrates for 3T3 cells when supplemented with 250 kD or 
with 35 kD proteins from rat CNS myelin as shown in FIG. 12. In these 
experiments, 250 kD and 35 kD proteins were added to amounts of liver (or 
sciatic nerve) protein equivalent to the ones of total CNS myelin protein 
from which they were prepared. We conclude that 35 kD and 250 kD proteins 
from rat CNS myelin act as inhibitors of neurite outgrowth and of 
fibroblast spreading, as their addition converts a neutral substrate into 
a nonpermissive one. 
7.2.4. Nonpermissive Substrate Property is Enriched in CNS White Matter and 
in Cultured Oligodendrocytes 
Considering the documented poor regenerative fiber growth found in mature 
CNS white matter (Nornes, H. A., et al., 1983, Cell Tissue Res. 230: 
15-35; Bjorklund, A. and Stenevi, U., 1984, Annu. Rev. Neurosci. 7: 
279-308), it was of particular interest to determine whether the 35 kD and 
250 kD neurite outgrowth-inhibiting proteins from CNS myelin were enriched 
in CNS white matter and in myelin forming cells. Protein-containing lipid 
vesicles from homogenates of different CNS regions were prepared and their 
substrate properties were determined. Rat CNS white matter material 
yielded highly nonpermissive liposomes containing inhibitory 250 kD and 35 
kD protein fractions (Table IV). 
TABLE IV 
______________________________________ 
DISTRIBUTION OF INHIBITORY 250 kD AND 35 kD 
PROTEIN FRACTIONS 
3T3 Spreading on Liposomes From: 
250 kD 35 kD 
Total Protein 
Fraction Fraction 
Protein Source 
(.mu.m.sup.2) 
(.mu.m.sup.2) 
(.mu.m.sup.2) 
______________________________________ 
CNS white matter 
211 .+-. 60 
158 .+-. 45 
242 .+-. 51 
CNS gray matter 
845 .+-. 106 
362 .+-. 65 
460 .+-. 55 
Optic nerve culture 
240 .+-. 67 
272 .+-. 52 
332 .+-. 58 
Sciatic nerve culture 
1,623 .+-. 173 
1,850 .+-. 250 
1,261 .+-. 141 
Trout CNS myelin 
1,050 .+-. 110 
1,150 .+-. 135 
1,585 .+-. 185 
______________________________________ 
*Protein (source) amounts were 100 .mu.g (total protein liposomes) and 50 
.mu.g (gelapplied protein). Sample preparation was as described supra in 
the Materials and Methods section. Tested protein, if not indicated 
otherwise, was obtained from rat tissues. 
Gray matter-derived liposomes contained markedly less nonpermissive 
activity. Significantly, high quantities of inhibitory activity were 
extracted from optic nerve-derived cell cultures. Such cultures contain 
highly nonpermissive, myelin marker-positive oligodendrocytes (see Section 
6.2.1., supra). Analogous protein fractions from a Schwann cell-containing 
culture yielded no inhibitory proteins. Thus, nonpermissive substrate 
activity in the nervous system, as detected by our assay, codistributes 
with CNS white matter and with myelin-forming oligodendrocytes. 
7.3. Discussion 
In this study, we have determined what makes rat CNS myelin a poor 
substrate. We first showed that brief treatment of the myelin with 
protease abolished nonpermissiveness, demonstrating the involvement of 
protein. These proteins require detergent to be separated from the myelin 
membranes. Solubilized myelin protein reconstituted with a 
phosphatidylcholine/phosphatidylserine mixture yielded liposomes with 
highly nonpermissive substrate properties. Liposomes with such unfavorable 
substrate properties were obtained from rat CNS myelin protein but not 
from the protein constituents of membrane fractions possessing permissive 
substrate properties (PNS myelin, liver). We, therefore, assume that 
nonpermissiveness is due to the same protein(s) in myelin and in 
myelin-derived liposomes. When myelin proteins were fractionated by 
SDS-PAGE, protein fractions with relative molecular masses of .about.35 kD 
and 250 kD were found to yield highly nonpermissive liposomes upon 
reconstitution. Furthermore, nonpermissive 35 kD and 250 kD protein 
fractions could not be found in rat PNS myelin nor in a liver-derived 
membrane fraction. Therefore, the presence of nonpermissive 35 kD and 250 
kD proteins and nonpermissive membrane fractions are correlated. Both 
protein fractions can function independently. 
We have determined that the presence of the 35 kD and 250 kD proteins from 
CNS myelin can be sufficient to generate a nonpermissive substrate by 
combining them with otherwise permissive substrate fractions. Thus, not 
only is nonpermissiveness of depleted rat CNS myelin restored (not shown), 
but supplemented liver or siatic nerve protein-derived liposomes become 
nonpermissive (FIG. 12). We therefore conclude that 35 kD and 250 kD 
proteins of rat CNS myelin are likely to be responsible for its 
nonpermissive substrate properties and that these proteins can be 
considered inhibitors of fibroblast spreading and of neurite outgrowth. 
Proof that the proteins are indeed the cause of CNS white matter 
nonpermissiveness was obtained by use of specific blocking antibodies; 
such antibodies neutralized the nonpermissiveness of gel-purified 
inhibitors-containing liposomes, of CNS myelin membranes, and of living 
cultured oligodendrocytes (as described in Section 8, infra). 
8. ANTIBODY AGAINST MYELIN-ASSOCIATED INHIBITOR OF NEURITE GROWTH 
NEUTRALIZES NONPERMISSIVE SUBSTRATE PROPERTIES OF CNS WHITE MATTER 
The examples described herein (Caroni and Schwab, March 1988, Neuron 1: 
85-96) demonstrate that an inhibitory substrate mechanism prevents 
neurites from growing into optic nerve explants in vitro, over living, 
cultured oligodendrocytes, and over myelin used as a culture substrate. 
CNS white matter from higher vertebrates and cultured differentiated 
oligodendrocytes are nonpermissive substrates for neurite growth and 
fibroblast spreading. Monoclonal antibodies, termed IN-2 and IN-1, were 
raised against 35 kD and 250 kD proteins respectively with highly 
nonpermissive substrate properties extracted from CNS myelin fractions. 
IN-1 and IN-2 bound both to the 35 kD and 250 kD inhibitors and to the 
surface of differentiated cultured oligodendrocytes. Adsorption of 
nonpermissive CNS myelin or nonpermissive oligodendrocytes with either 
antibody markedly improved their substrate properties. Optic nerve 
explants injected with IN-1 or IN-2 allowed axon ingrowth of cocultured 
sensory and sympathetic neurons. We conclude that the nonpermissive 
substrate properties of CNS white matter are due to these membrane 
proteins on the surface of differentiated oligodendrocytes and to their in 
vivo product, myelin. 
8.1. Experimental Procedures 
8.1.1. Cell Culture 
Mouse NIH 3T3 cells were cultured and assayed for spreading behavior in 
DMEM containing 10% FCS. SCGs from newborn rats were dissected and 
dissociated into single cells as described (Mains and Patterson, 1973, J. 
Cell. Biol. 59: 329-345; Schwab and Thoenen, 1985, J. Neurosci. 5: 
2415-2423). Neurons were cultured in an enriched L15 medium (Mains and 
Patterson, supra) with 5% rat serum and 100 ng/ml of 2.5S NGF. Overgrowth 
by non-neuronal cells was prevented by inclusion of cytosine arabinoside 
(10.sup.-5 M) in the culture medium. Inhibitory oligodendrocyte-containing 
cultures were prepared from the optic nerves of 8-10 day old rats as 
described (see Section 6.1.1., supra). Optic nerve cultures were 
maintained in an enriched L15 medium with 5% rat serum. P3U myeloma cells 
and their hybridomas were cultivated in Iscove medium supplemented with 
glutamine, antibiotics, 10.sup.-4 M .beta.-mercaptoethanol, and 10% human 
serum. 
8.1.2. Substrate Preparation 
Myelin fractions were isolated as described in Section 6.1.5., supra). 
Briefly, carefully cleaned adult rat spinal cord tissue (CNS myelin) or 
rat sciatic nerve (PNS myelin) was homogenized in isotonic sucrose buffer 
with a polytron homogenizer, and myelin membrane fractions were obtained 
by flotation of low speed supernatants to densities below that of 0.85M 
sucrose (Colman, et al., 1982, J. Cell Biol. 95: 598-608). All isolation 
media contained trasylol (100 U/ml), 5 mM iodoacetamide, and 5 mM EDTA to 
reduce protease digestion (this reagent mixture is designated herein as 
protease inhibitors). Finally, myelin fractions were washed hypotonically 
in 30 mM Hepes (pH 7.4) (medium A) plus protease inhibitors and frozen in 
aliquots at -80.degree. C. To prepare protease-digested myelin membranes, 
the myelin was washed in medium A without protease inhibitors and 
subsequently incubated at a concentration of 1 mg/ml in the presence of 
0.1% trypsin (Sigma). After 10 minutes at room temperature, 0.2% trypsin 
inhibitor (Sigma) was added and membranes were washed free of protease in 
medium A. Oxidative chemical deglycosylation of myelin membranes was 
performed by the periodate method as described (Beeley, 1985, in 
Laboratory Techniques in Biochemistry and Molecular Biology, R. H. Burdon 
and P. H. Van Knippenberger, eds., Elsevier, Amsterdam, pp. 279-288). 
Liposomes containing myelin membrane proteins were prepared by the cholate 
dialysis method (see Section 7.1.4, supra). Briefly, solubilized protein 
was precipitated in a 10-fold excess volume of ice-cold acetone. 
Precipitated protein was collected by centrifugation after a 15 hour 
incubation at 4.degree. C. and resuspended in 2.5% cholate in medium A. A 
5- to 50-fold excess (v/v) of phospholipids 
(phosphatidylcholine/phosphatidylserine, 10:1) in medium A plus 2.5% 
cholate was then added to the solubilized protein. The lipid-protein 
mixture was applied to a Sephadex G50 column equilibrated in medium A and 
liposomes were collected in the void volume. When membrane fractions were 
used as protein source, precipitated protein was resuspended at 
approximately 1 mg/ml, while gel-extracted protein was resuspended at 
approximately 50 .mu.g/ml. 
Gel extraction of inhibitory proteins from CNS myelin was performed as 
described (see Section 7.1.5, supra). Myelin protein was fractionated on 
3%-15% gradient gels under reducing conditions. For this purpose, samples 
were preincubated for 30 minutes at room temperature in SDS-PAGE sample 
buffer containing 2% SDS and .beta.-mercaptoethanol. Protein from gel 
regions to be analyzed was extracted in the presence of 0.5% SDS and 50 
.mu.g/ml insulin (Sigma). The latter was included to reduce losses due to 
adsorption of protein from low concentration solutions. The added insulin 
was removed from the protein to be tested by the Sephadex G50 column 
(liposome formation procedure). When gel-extracted protein was used as 
immunogen, no insulin was included in the gel extraction medium. 
8.1.3. Immunological Methods 
The following antisera and monoclonal antibodies were used in this study; 
anti-N-CAM (neural cell adhesion molecule) antiserum (gift of M. 
Schachner, Heidelberg, FRG), anti-tenascin antiserum (gift of R. 
Chiquet-Ehrismann, Basel, Switzerland), anti-J1 antiserum (gift of M. 
Schachner), monoclonal antibodies O.sub.4 (anti-sulfatide) and O.sub.1 
(anti-galactocerebroside) (gifts of M. Schachner). 
Anti-CNS myelin antiserum was produced in rabbits by the injection of 200 
.mu.g of myelin protein per immunization step. The cold-soluble fraction 
of the antiserum was heat-inactivated by incubation at 56.degree. C. for 1 
hour. 
To produce anti-inhibitory substrate monoclonal antibodies, BALB/c mice (6 
week old females) were injected with approximately 50 .mu.g of 
gel-extracted inhibitory (35 kD or 250 kD) fraction from rat CNS myelin. 
Gel-extracted protein was precipitated in acetone and resuspended at 1 
mg/ml in sterile PBS plus 0.1% cholate. Mice were immunized twice at 3 
week intervals, sera were tested for production of 
nonpermissiveness-neutralizing antibodies (see also below), and mice with 
strong neutralizing sera were used for hybridoma production. 
8.1.3.1. Radioimmunoassay 
Antibody presence was detected by a solid phase radioimmunoassay (Carlson 
and Kelly, 1983, J. Biol. Chem. 258: 11082-11091) using .sup.125 I-labeled 
goat anti-mouse antibody (Bio-Rad) as a probe. 
Wells of 96 well plates were coated by exposing them to appropriate antigen 
(2 .mu.g of protein per ml of 30 mM Tris pH 7.4!, 160 mM NaCl 
Tris-saline!; 50 .mu.l per well) for at least 3 hours at room 
temperature. Coated wells were then washed in Tris-saline plus 1% BSA, 
incubated in the presence of the hybridoma supernatants, and finally 
assayed for the presence of bound antibody with .sup.125 I-labeled goat 
anti-mouse antibody (approximately 10.sup.5 cpm per well). Values obtained 
with different antigens cannot be compared quantitatively, as adsorption 
to the wells varied among different antigens. Background values for goat 
anti-mouse binding in the absence of mouse antibodies were routinely 
subtracted. Signals of less than 2 times background values (100-150 cpm) 
were considered nonsignificant. 
8.1.3.2. Immunoblots 
Transfer of CNS myelin protein fractionated by SDS-PAGE onto nitrocellulose 
was performed in 50 mM sodium phosphate buffer (pH 5.5), 2 mM EDTA, 0.05% 
SDS (Filbin, M. T. and Poduslo, S. E., 1986, Neurochem. Int. 9: 517-520). 
Transfer time was 3 hours at 1.6 A. Incubation procedures with antibodies 
and .sup.125 I-labeled second antibody followed standard procedures. 
Antibody-binding protein bands were visualized by autoradiography using 
high sensitivity Kodak X-ray films (X-OMAT). 
Preparation of cell cultures for immunofluorescence microscopy was 
performed as follows. Cultures were rinsed in PBS and pre-fixed at 
37.degree. C. with fixation medium containing 4% paraformaldehyde. Upon 
extensive rinsing with PBS, cultures were incubated with the first 
antibody for 45 minutes at room temperature (antibody dilutions, 1:50 for 
antisera and ascites; 1:3 for hybridoma supernatants; dilution buffer 
consisted of isotonic, sucrose-containing phosphate buffer (pH 7.4) plus 
5% BSA). Unbound antibody was removed with 5% BSA-containing medium. After 
thus staining the cells with monoclonal antibodies, the cells were 
incubated with a 1:50 dilution (in 5% BSA-containing medium) of the second 
antibody, a rabbit anti-mouse antibody (SAKO, Copenhagen, Denmark) in 5% 
BSA-containing medium. The rabbit anti-mouse incubation enhanced signal 
intensities and was performed for 30 minutes at room temperature. Cells 
were then fixed in isotonic buffer containing 4% paraformaldehyde. 
Fixation was interrupted after 30 minutes. Upon incubation with 
appropriate fluorescently labeled second antibody and subsequent washes in 
dilution buffer, bound antibody was visualized on an Olympus Vanox 
fluorescence microscope. Control experiments with hybridoma medium were 
performed to exclude nonspecific fluorescent signals. Highly branched 
oligodendrocytes or HBOs were identified by their characteristic 
morphology and by double labeling experiments with the antibody O.sub.1 
(see Section 6.1.3 supra). 
Laminin was visualized by indirect immunofluorescence using a rabbit 
antiserum against EHS-tumor laminin on frozen sections of freshly 
dissected adult rat optic and sciatic nerves and on sections of nerves 
after 4 weeks in culture (see below). 
Immunoprecipitation of IN-1-binding (and of IN-2-binding) proteins was 
performed in immunoprecipitation buffer consisting of 150 mM NaCl, 30 mM 
Hepes (pH 8.2), 2% NP40.TM., 0.5% sodium deoxycholate, plus protease 
inhibitors. Antibody was bound either to solubilized CNS myelin protein 
(solubilization in immunoprecipitation buffer for 1 hour at 4.degree. C.) 
or to intact myelin membranes. In both cases, 100 .mu.g of myelin protein 
was incubated with 1 ml of hybridoma supernatant for 1 hour at room 
temperature. Myelin membranes incubated in the presence of antibody were 
washed twice in medium A and solubilized in immunoprecipitation buffer. 
Rabbit anti-mouse was added in both immunoprecipitation protocols to 
solubilized antigen-antibody complex (20 .mu.g of rabbit anti-mouse per ml 
of hybridoma supernatant), and incubation was allowed to proceed for an 
additional 1 hour period at room temperature. Antigen-antibody-rabbit 
anti-mouse complex was finally sedimented with S. aureus cells (Sigma). 
Elution was performed by boiling for 5 minutes in 100 mM ammonium chloride 
(pH 11.5) plus 0.5% SDS and .beta.-mercaptoethanol. This procedure 
irreversibly inactivated present antibodies. Eluted, neutralized antigen 
was precipitated with acetone and assayed for its substrate properties 
upon reconstitution into liposomes. 
8.1.4. Substrate Testing Procedures 
Substrates were tested as described in Section 7.1.2 supra). Myelin 
fractions or liposomes in medium A were dried onto the wells of 
polylysine-coated Greiner dishes (Greiner, Murtingen, FRG). Unbound 
membranes were removed by 3 washes in Ca.sup.2+ - and Mg.sup.2+ -free 
Hank's medium, and substrate-testing cells, i.e., 3T3 fibroblasts or 
superior cervical ganglian SCG neurons, were immediately added to the 
dishes. When substrates were tested in the presence of antibody, bound 
substrates were incubated in the presence of undiluted hybridoma 
supernatants or in the presence of 1:30 dilutions (in Hank's medium) of 
antisera. After 15 minutes at 37.degree. C., four-fifths of the 
antibody-containing medium was removed and substituted with 
cell-containing medium. An analogous preincubation procedure was used in 
experiments aimed at testing HBO nonpermissiveness. 3T3 experiments were 
usually scored after a culture period of 1 hour, whereas SCG neurons were 
allowed to grow processes for 24 hours in culture. When 3T3 cells were 
preincubated with hybridoma supernatant, incubations were performed in 
gently agitated suspensions for a period of 15 minutes at room 
temperature. Cells were then sedimented, resuspended in culture medium, 
and added to substrate-adsorbed Greiner dish wells. 3T3 cells were added 
at a density of 30,000 cells per cm.sup.2, and SCG neurons were added at a 
density of 20,000 cells per cm.sup.2. 
Quantitative evaluation of spreading was performed with a surface 
integration program on at least 30 cells per experimental point. 
Photographs of randomly selected fields were used for this purpose, and 
the outlines of all spread cells present in the field were traced manually 
with a graphic stylus connected to a computer. Only spread cells were 
considered, and a zero-spreading value (strongly refractory and round 
cells) was subtracted. Experiments were found to be subject to only small 
quantitative variations, and values from representative experiments are 
given. Spreading degrees are given as means plus or minus standard error 
of the mean. 
Quantitative evaluation of HBO inhibition of fibroblast spreading was 
performed by determining areas of overlap between O.sub.1.sup.+ 
oligodendrocytes and 3T3 fibroblasts 1-2 hours after the addition of 3T3 
cells to a 2 day old optic nerve culture. The ratio of 3T3-oligodendrocyte 
overlap to the total area of oligodendrocytes was compared with the 
proportion of the total examined culture area occupied by 3T3 cells. Zero 
inhibition was defined as the absence of apparent discrimination by 
spreading fibroblasts against surface occupied by oligodendrocytes. 
8.1.5. Neurite Growth into Optic Nerve Explants in vitro 
Optic nerves of young adult rats were cultured together with dissociated 
rat sympathetic or sensory neurons as previously described (Schwab and 
Thoenen, 1985, J. Neurosci. 5: 2415-2423). Briefly, optic nerves were 
rapidly dissected from 6-8 week old female rats, cleaned from adhering 
meninges, and injected from both sides using a 10 .mu.l Hamilton syringe 
with 2 .mu.l of either IN-1 or IN-2 hybridoma supernatant or (controls) 
with O.sub.1 hybridoma supernatant or antibody-free hybridoma medium. A 
Teflon ring with silicon grease separated the three chambers, and optic 
nerves were placed through the silicon grease, reaching from the middle 
chamber into one or the other side chamber. The respective antibodies were 
present in the side chambers at a dilution of 1:10 throughout the culture 
period. Dissociated newborn rat SCG or dorsal root ganglion neurons were 
plated in the central chamber in L15 medium with rat serum and NGF, thus 
having access to one end of both nerves. After 3 weeks in culture, 
cultures were fixed with 2.5% glutaraldehyde and disassembled. The nerve 
explants were separately embedded in EPON. Sections for electron 
microscopy were cut from three regions of each nerve; within the first 1 
mm in the central chamber, from the region of the nerve under the Teflon 
ring, and at a distance of 3 mm from the central chamber end of the nerve 
(side chamber region). Most of the sections comprised the entire nerve in 
cross sections; they were systematically screened for the presence of 
axons using electron microscopy. 
8.2. Results 
8.2.1. Antiserum Against Myelin Neutralizes the Nonpermissive Substrate 
Effects of CNS Myelin and of HBOs 
Antisera were generated against rat CNS myelin and adsorbed to 
polylysine-bound myelin. The antibody-adsorbed myelin was then assayed for 
its substrate properties in supporting fibroblast spreading. The antiserum 
contained antibodies that neutralized CNS myelin nonpermissiveness. 
Nonimmune rabbit serum did not significantly modify myelin substrate 
properties. In these experiments, care was taken to, first, 
heat-inactivate rabbit serum fractions in order to prevent highly toxic 
complement reaction and, second, to deplete the same fractions of 
cold-insoluble proteins, which included fibronectin as a strong promoter 
of fibroblast spreading and attachment. The antiserum was also very 
effective in neutralizing HBO nonpermissiveness. Neutralization was 
specific, as rabbit antiserum against tenascin (Chiquet-Ehrismann et al., 
1986, Cell 47: 131-139) (FIGS. 14A-F), N-CAM (neural cell adhesion 
molecule), and J1 (Kruse et al., 1985, Nature, 316: 146-148) did not 
influence myelin or HBO substrate properties. The cell adhesion molecules 
N-CAM and J1 were present in substantial amounts on the surface of HBOs as 
detected by immunofluorescence. Tenascin antigenicity was absent from the 
myelin (shown by radioimmunoassay) as well as from the HBO surface (shown 
by immunofluorescence). This finding is important as it demonstrates that 
the documented, unfavorable substrate properties of tenascin 
(Chiquet-Ehrismann et al., 1986, Cell 47: 131-139) are not responsible for 
myelin or HBO substrate properties. The experiments with myelin-antiserum 
demonstrated that the properties of both tested substrates, myelin and 
oligodendrocytes, could be improved by antibody binding. 
8.2.2. IN-1: A Monoclonal Antibody Against Gel-Purified 250 kD Inhibitor 
Fraction from CNS Myelin Neutralizes Myelin Nonpermissiveness 
Mice were immunized with rat CNS myelin 250 kD protein fraction, hybridomas 
were raised, and supernatants were screened for anti-myelin antibodies. 
Positives were rescreened for neutralization of CNS myelin 
nonpermissiveness by the 3T3 cell spreading assay. Five myelin-positive 
antibodies fulfilled the second screening criterion to varying degrees. 
The antibody with the strongest neutralizing properties was selected and 
designated IN-1. 
Adsorption of liposomes containing the 250 kD protein, liposomes containing 
the 35 kD protein, and rat CNS myelin with IN-1 drastically reduced 
nonpermissiveness in all three cases (FIGS. 13A-H). Neutralization was 
slightly less efficent for CNS myelin membranes (FIGS. 13A and 13D), 
possibly due to incomplete saturation of inhibitory sites by the antibody. 
The antibody bound to inhibitor-containing liposomes and to CNS myelin, 
but not to PNS myelin (Table V). 
TABLE V 
______________________________________ 
IN-1 BINDS TO 35 kD AND 250 kD 
MEMBRANE PROTEINS FROM RAT CNS MYELIN* 
Amount of Antibody Bound 
(cpm of .sup.125 I-Labeled Goat Anti-Mouse) 
Antigen IN-1 lG9** O1* 
______________________________________ 
CNS myelin: 
Control 550 .+-. 35 
1500 .+-. 110 
10850 .+-. 550 
Trypsin-treated 
25 .+-. 20 
40 .+-. 30 
8200 .+-. 480 
PNS myelin: 80 .+-. 25 
350 .+-. 30 
9200 .+-. 500 
Liposomes containing: 
250 kD CNS myelin protein 
250 .+-. 30 
45 .+-. 20 
80 .+-. 50 
35 kD CNS myelin protein 
350 .+-. 35 
40 .+-. 30 
90 .+-. 60 
______________________________________ 
*Antibody binding sites were detected by a solid phase radioimmunoassay 
using .sup.125 Ilabeled goat antimouse antibody as a probe. Liposomes wer 
prepared from gelextracted CNS myelin protein (100 .mu.g of myelin protei 
added to the gel). Values are given after subtraction of background 
binding in the absence of primary antibody. Background values for 
liposomes were essentially identical to the ones obtained with antibody i 
the presence of proteinfree liposomes, i.e., approximately 120-150 cpm. 
**Antibody lG9 is an antimyelin monoclonal antibody that binds to the 
surface of differentiated oligodendrocytes and to protein of 110 kD on 
Western blots of rat CNS myelin. 
Neutralization of inhibitory substrate properties of myelin fractions was 
observed for superior cervical ganglion (SCG) neurons (FIGS. 13A-H), for 
3T3 fibroblasts (see Table VI), and for neuroblastoma cells in the 
presence of dibutyryl-cAMP. 
TABLE VI 
______________________________________ 
IN-1 NEUTRALIZES NONPERMISSIVENESS OF CNS MYELIN 
AND ITS SPREADING INHIBITORS OF 35 kD AND 250 kD* 
3T3 Spreading (.mu.m.sup.2 ) 
Substrate Control +IN-l O1 
______________________________________ 
CNS myelin 278 .+-. 31 1446 .+-. 114 
250 .+-. 36 
250 kD liposomes 
213 .+-. 11 1335 .+-. 151 
245 .+-. 35 
35 kD liposomes 
185 .+-. 18 1286 .+-. 113 
210 .+-. 21 
Protein-free liposomes 
1520 .+-. 145 
1410 .+-. 105 
1495 .+-. 145 
______________________________________ 
*Spreading extents were estimated after 1 hour in culture in the presence 
or the absence of hybridoma supernatant. Substrate protein amounts 
adsorbed to the wells of Greiner dishes were as follows: CNS myelin, 20 
.mu.g per cm.sup.2 ; liposomes, 100 .mu.g of CNS myelin protein were 
applied to the gel lane from which proteins of indicated apparent 
molecular weight were extracted, and the entire extracted and 
reconstituted protein was applied to the culture well. Apparent molecular 
weight ranges were estimated with molecular weight standards (BioRad) and 
were about 35 .+-. 3 kD and 250 .+-. 15 kD, respectively. 
In control experiments, preadsorption of the fibroblasts with antibody did 
not modify their behavior on antibody-free CNS myelin. Also, IN-1 had no 
influence on 3T3 spreading when these cells were seeded onto a glass 
surface, on a protein-free lipid vesicle, or on permissive liposomes 
containing peripheral 250 kD protein (see FIGS. 13A-H). In these latter 
cases, cell attachment and spreading were slightly impaired, but no 
improvement was obtained with IN-1, demonstrating the specificity of the 
antibody effect for neutralizing myelin-derived inhibitors. Neutralization 
was due to the antibody fraction in IN-1-containing supernatants, as an 
ammonium sulfate-precipitated fraction of IN-1 ascitic fluid was equally 
effective. 
IN-1 binding is completely abolished by a brief pretreatment of the myelin 
with trypsin, demonstrating that the antibody does not bind to glycolipids 
(Table V). IN-1 antibody when preadsorbed onto HBO-containing cultures 
also efficiently reduced HBO nonpermissiveness (see FIGS. 14A-F). In 
control experiments, 3T3 cells never invaded more than 10% of the surface 
of O.sub.1.sup.+ (galactocerebroside; marker for differentiated 
oligodendrocytes) HBOs. Fibroblasts seeded directly onto HBOs failed to 
spread and eventually detached. Often more than 50% of HBO surface was 
covered by fibroblasts in the presence of IN-1. In addition, spreading of 
fibroblasts on antibody-adsorbed HBOs was frequently observed (FIGS. 
14A-F). Interestingly, quantitative determination of the substrate 
properties of HBOs in the presence of IN-1 showed that 3T3 cells prefer 
HBOs over the polylysine-coated culture dish under this condition (FIGS. 
14A-F). This behavior could be related to the presence of cell adhesion 
molecules like myelin associated glycoprotein, J1, or N-CAM on these 
oligodendrocytes. FIG. 16 also shows that IN-1 bound to the surface of 
living HBOs. Specific staining of intact cells with the morphology of 
astrocytes, fibroblasts or immature A.sub.2 B.sub.5.sup.+ oligodendrocytes 
was not detected by our method. Also, no specific IN-1 staining could be 
detected on the surface of living neuronal cells or neuroblastoma cells. 
The observed weak staining of HBOs was probably due to the fact that 
spreading inhibitors are minor proteins in myelin and optic nerve culture 
fractions. These experiments demonstrate that nonpermissiveness in HBOs 
is, as previously shown for myelin, an IN-1-affected process. 
8.2.3. 250 kD and 35 kD Inhibitors from CNS Myelin Share Two Neutralizing 
Epitopes 
As shown in Table VI, IN-1 did bind to liposomes containing the 35 kD 
inhibitor. Therefore, the epitope defined by IN-1 is shared between the 
250 kD and 35 kD inhibitors (see also FIG. 16). Such an epitope may be a 
polypeptide since treatment of the myelin with periodate to remove 
carbohydrate did not affect IN-1 binding. Table VI demonstrates that the 
35 kD inhibitor was neutralized by IN-1. As the antibody neutralized the 
inhibitory substrate properties of myelin membranes, these experiments are 
consistent with the interpretation that both the 250 kD and the 35 kD 
inhibitor contribute to myelin nonpermissiveness. 
Control experiments (Table VI) excluded that IN-1 neutralization was due to 
nonspecific masking of the myelin as a consequence of antibody binding, as 
monoclonal antibodies O.sub.1 and O.sub.4, which bind to very abundant 
antigens on the surface of myelin and HBOs, did not reduce the 
nonpermissive substrate effects of either myelin (Table VI, for O.sub.1) 
or living HBOs (FIG. 15, for O.sub.4). 
Immunization experiments, as previously described for the 250 kD protein, 
were performed with the gel-purified 35 kD inhibitor fraction from rat CNS 
myelin, confirming the relatedness of the 250 kD and 35 kD myelin 
inhibitors. Hybridomas produced from such mice were tested as described 
for monoclonal antibody IN-1. The strong neutralizing antibody IN-2 was 
selected. Neutralization and binding properties of IN-2 are summarized in 
Table VII. 
TABLE VII 
______________________________________ 
IN-2 BINDS TO CNS MYELIN INHIBITORY SUBSTRATES 
AND NEUTRALIZES THEIR NONPERMISSIVE 
SUBSTRATE PROPERTIES* 
IN-2 Binding 
(.sup.125 I-Labeled Goat 
3T3 Spreading (.mu.m.sup.2) 
Antigen/Substrate 
Anti-Mouse cpm) 
No IN-2 +IN-2 
______________________________________ 
CNS myelin 975 245 .+-. 15 
1300 .+-. 121 
CNS myelin, 100 1485 .+-. 110 
1430 .+-. 158 
trypsin-treated 
CNS myelin liposomes 
700 180 .+-. 18 
1040 .+-. 110 
35 kD liposomes 
285 215 .+-. 15 
1250 .+-. 120 
250 kD liposomes 
410 205 .+-. 11 
1180 .+-. 108 
Protein-free liposomes 
100 1385 .+-. 125 
1420 .+-. 160 
______________________________________ 
*Experimental details are described supra in Experimental Procedures and 
Tables V and VI. Data presented in the table were obtained with a 1:100 
dilution (in PBS) of ammonium sulfateprecipitated antibody from ascitic 
fluid (10 .mu.g of protein per ml after dilution). CNS myelin liposomes 
were formed from 20 .mu.g of CNS myelin protein. 
Thus, IN-2 bound to both the 35 kD and the 250 kD protein, it neutralized 
nonpermissiveness of myelin membranes and HBOs, and it bound to the 
surface of living HBOs. IN-1 and IN-2 epitopes are not identical: IN-2, 
but not IN-1, strongly bound to cytoskeleton-associated antigens when 
astrocytes or fibroblasts were permeabilized. As found for IN-1, IN-2 did 
not bind to protease-treated myelin. 
8.2.4. IN-1 Specifically Immunoprecipitates Nonpermissive Substrate 
Activity from Solubilized Myelin Protein 
Since the inhibitory protein fractions used in this study apparently 
contained more than one protein species, immunoprecipitation experiments 
were performed to determine whether IN-1 binds directly to neurite growth- 
and fibroblast spreading-preventing protein(s). 
Solubilized myelin protein was adsorbed with IN-1 antibody, and 
antigen-antibody complex was sedimented with rabbit anti-mouse bound to 
Staphylococcus aureus cells. Antigen-antibody complexes were then 
dissociated under denaturing and reducing conditions to irreversibly 
inactivate the IN-1 antibody. Liposomes formed in the presence of 
immunoprecipitated protein were highly inhibitory for fibroblast spreading 
(Table VIII). 
TABLE VIII 
______________________________________ 
IN-1 SPECIFICALLY REMOVES INHIBITORY SUBSTRATES 
FROM CNS MYELIN PROTEIN, INHIBITORY PROTEINS 
OF 35 kd AND 250 kd ARE IMMUNOPRECIPITATED BY IN-1* 
3T3 Spreading (.mu.m.sup.2) 
Protocol 1 
Protocol 2 
______________________________________ 
Myelin Protein Fraction 
Total 322 .+-. 32 
255 .+-. 25 
IN-1-depleted 1361 .+-. 61 
1280 .+-. 90 
O.sub.1 -depleted 
612 .+-. 63 
ND 
IN-1-immunoprecipitated 
218 .+-. 16 
215 .+-. 18 
O.sub.1 -immunoprecipitated 
1218 .+-. 108 
1410 .+-. 128 
From IN-1-precipitated: 
250 kD liposomes ND 245 .+-. 18 
35 kD liposomes ND 270 .+-. 21 
______________________________________ 
*Details of immunoprecipitation protocols are given in Section 8.1.3.2., 
supra. 
Protocol 1: immunoprecipitation from solubilized CNS myelin protein. 
Protocol 2: immunoprecipitation upon binding of antibody to intact myelin 
membranes and subsequent solubilization of antigenantibody complexes. In 
both protocols, immunoprecipitation was performed using 100 .mu.g of CNS 
myelin protein. Total refers to liposomes formed from 1% of the starting 
material. Liposome fractions designated depleted were formed from 1% of 
the immunoprecipitation supernatant. Those designated immunoprecipitated 
were formed from 5% of the immunoprecipitated and eluted material. 
Finally, in the experiments presented in the second part of the table, 
IN1-immunoprecipitated protein (from 500 .mu.g of starting CNS myelin 
protein) was separated by SDSPAGE, and gel regions of indicated apparent 
molecular weight were extracted from the gel and reconstituted as 
described in Section 8.1.3.2., and previous tables. In these cases, 
liposomes supra corresponding to 5% of the gelextracted protein were 
adsorbed to the 1 cm.sup.2 wells. 
ND, not determined. 
In control experiments, both O.sub.1 antibody (Table VIII) and a monoclonal 
antibody against 110 kD myelin protein did not immunoprecipitate 
inhibitory substrate. 
Immunoprecipitation of inhibitory substrate with IN-1 could also be 
performed when antibody was first bound to myelin membranes (Table VIII). 
In the latter case, membranes were subsequently washed free of unbound 
antibody, myelin protein was solubilized, and antigen-antibody complex was 
sedimented as described above. This experiment demonstrated that the 
inhibitory substrate-associated IN-1 epitope(s) was accessible to antibody 
in its native myelin membrane location. When immunoprecipitated proteins 
were subsequently separated by SDS-PAGE, inhibitor-containing protein 
fractions of 35 kD and of 250 kD could be extracted from the gel (Table 
VIII). Therefore, 35 kD and 250 kD inhibitors of neurite growth and 
fibroblast spreading expose the IN-1 epitope on the surface of myelin 
membranes. 
FIG. 16 shows that IN-1 binding to CNS myelin proteins fractionated by 
SDS-PAGE and adsorbed onto nitrocellulose was restricted to a subset of 
minor myelin proteins. While binding to 250 kD protein was consistently 
observed, binding to protein in the 35 kD region was weak and often not 
detectable. In addition, IN-1 bound to 56 kD protein, which had not 
previously been recognized as yielding inhibitory liposomes upon 
reconstitution. When myelin fractions yielding strong IN-1.sup.+ 56 kD 
protein were used as the source of gel-purified 56 kD protein, highly 
inhibitory liposomes were obtained upon reconstitution. While essentially 
no binding of IN-1 to intact, permissive PNS myelin membranes could be 
observed (Table V), Western blot analysis of PNS myelin protein with IN-1 
revealed immunoreactive material of 300-400 kD. Upon gel extraction and 
reconstitution, this latter material yielded liposomes with permissive 
substrate properties. Masking of a hypothetical inhibitory substrate by 
highly favorable substrate from the same 300-400 kD PNS myelin protein 
region cannot be excluded. However, attempts to immunoprecipiate 
inhibitory substrate from solubilized PNS myelin protein with IN-1 have 
failed. It therefore seems reasonable to assume that the antibody IN-1 
also binds to proteins with no inhibitory substrate properties. Therefore, 
identification of inhibitory substrates by Western blot analysis with the 
antibody IN-1 is presently not warranted. 
8.2.5. Nonpermissiveness of Adult Optic Nerve is Neutralized by Adsorption 
with IN-1 Antibody 
Optic nerve explants, in contrast to sciatic nerve explants, have been 
observed not to support growth of neurites in vitro, even when optimal 
amounts of appropriate neurotrophic factor was present (Schwab and 
Thoenen, 1985, J. Neurosci. 5: 2415-2423). Cultured optic nerve explants 
were assayed for laminin immunoreactivity as neurite growth is known to be 
supported by laminin and furthermore since laminin is known to be present 
in sciatic nerve but not in optic nerve in situ. Laminin was exclusively 
present on the pial basement membrane and around blood vessels when 
freshly dissected optic nerve from adult rat was analyzed. The explant, 
however, contained substantial amounts of strongly laminin-positive cells, 
presumably astrocytes, after 3-4 weeks in vitro (FIGS. 17A-B). Despite the 
presence of laminin, no neurites were found to grow into optic nerve after 
periods of up to 5 weeks in vitro. These findings supported the 
interpretation that a nonpermissive substrate present in the optic nerve 
explants is responsible for its unfavorable microenvironment. 
In subsequent experiments, IN-1 antibody was injected into the optic nerve 
explant prior to insertion of the explant in a three-compartment chamber 
culture system (Schwab and Thoenen, 1985, J. Neurosci. 5: 2415-2423). In 
addition, IN-1-containing supernatant was also added to the compartment 
containing the distal end of the nerves for the duration of the 
experiment. In control experiments, supernatants rich in O.sub.1 antibody 
were injected and included in the culture medium. 
The results from these experiments demonstrated that IN-1, but not O.sub.1 
antibody, effectively promoted extensive growth of sympathetic and sensory 
neurites into the optic nerves (Table IX, FIGS. 18A-D). 
TABLE IX 
______________________________________ 
INJECTION OF OPTIC NERVE EXPLANTS WITH ANTIBODY 
IN-1 RESULTS IN INGROWTH OF AXONS INTO OPTIC 
NERVE IN VITRO* 
Antibody IN-1 Antibody O.sub.1 
Optic Region Optic Region 
Culture 1 mm 3 mm 1 mm 3 mm 
______________________________________ 
1 +++ +++ ++ - 
2 ++ +++ + + 
3 ++ +++ + + 
4 ++ ++ + - 
5 +++ +++ 
6 ++ ++ - - 
______________________________________ 
*Optic nerve explants were injected with antibody IN1 or antibody O.sub.1 
and then placed into chamber cultures with sensory neurons in the central 
chamber. After 3 weeks in culture, nerves were systematically examined by 
electron microscopy. Presence of axons at 1 and 3 mm in the optic nerves 
of representative experiment. + indicates 1-20 axons; ++: 20-50 axons; 
+++: &gt;50 axons per cross section. Large numbers of axons were found deep 
in the IN1-injected nerves, but not in the O.sub.1 -injected nerves. 
Neurites extended for lengths of more than 3 mm into optic nerves in the 
presence of IN-1 (Table IX). Although preferred as a substrate, growth was 
not restricted to regions adjacent to basal membrane, and contact of 
ingrowing neurites with myelin could frequently be observed (FIGS. 18A-B). 
In some experiments, damaged control nerves with large tissue-free spaces 
did allow limited neurite growth. In those cases, however, neurites were 
not found in contact with myelin sheets. Neurite growth over a distance of 
3 mm into the optic nerves was observed in 5 out of 6 cases when IN-1 was 
present. Growth in O.sub.1 -containing nerve was observed in 1 out of 5 
cases (Table IX). 
These findings strongly suggest that nonpermissiveness of optic nerve 
explants in vitro is due to IN-1-binding inhibitory substrate. As 35 kD 
and 250 kD inhibitory substrates from CNS myelin are found in optic nerve 
tissue, these proteins are likely to be responsible for its nonpermissive 
microenvironment in vitro and possibly also in vivo. 
8.3. Discussion 
The experiments described herein demonstrate that monoclonal antibodies 
raised against each gel-purified inhibitor fraction neutralized or greatly 
reduced the nonpermissiveness of both inhibitors, of isolated myelin 
membrane fractions, of cultured HBOs, and of adult rat optic nerve 
explants. The antibodies bind to the surfaces of myelin membranes and 
cultured oligodendrocytes. They specifically immunoprecipitate inhibitory 
substrate proteins of 35 kD and 250 kD from myelin protein fractions. We 
conclude that nonpermissiveness of adult CNS white matter-derived tissues, 
cells, and subcellular fractions is due to the same inhibitory substrate 
mechanism involving IN-1-binding (and IN-2-binding) proteins. Clearly, 35 
kD and 250 kD inhibitors share two antigenic sites, IN-1 and IN-2. In both 
cases, antibody binding abolished nonpermissive substrate properties. Our 
data are consistent with the interpretation that the proteins reponsible 
for adult CNS white matter nonpermissiveness are the 35 kD and 250 kD (and 
56 kD) inhibitory substrates extracted from rat CNS myelin. 
9. INVOLVEMENT OF A METALLOPROTEASE IN GLIOBLASTOMA INFILTRATION INTO 
CENTRAL NERVOUS SYSTEM TISSUE IN VITRO 
In the examples detailed herein, we describe a membrane-associated 
metalloprotease which plays a crucial role in the malignant tumor 
infiltration of CNS tissue in vitro by the rat glioblastoma cell line C6. 
We have discovered that malignant tumor infiltration of CNS tissue in vitro 
by the glioblastoma line C6, requires a plasma membrane bound 
metallodependent degradative activity. C6 cells infiltrate optic nerve 
explants, attach and spread on white and grey matter of cerebellar frozen 
sections or on CNS myelin. The metal ions chelator 1,10-phenanthroline and 
the dipeptide cbz-tyr-tyr, but not inhibitors for three other classes of 
proteases, blocked up to 67% of C6 cell spreading on CNS myelin. A 
metallodependent activity neutralizing CNS myelin inhibitory substrate 
properties toward 3T3 cells, is associated with a C6 plasma membrane 
fraction. The same inhibitors of metalloprotease also impaired 
infiltration of CNS nerve explants and spreading on the CNS white matter 
of cerebellar frozen sections. 
9.1. Materials and Methods 
9.1.1. Cell Cultures 
Rat C6, mouse NIH 3T3 and B16 cells were cultured in Dulbecco's modified 
Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 
usually to maximally 70-80% confluency. Cells were harvested with a short 
trypsin treatment (0.1% in Ca.sup.2+ /Mg.sup.2+ -free Hank's medium for 90 
seconds) stopped by addition of FCS in excess, collected by 
centrifugation. Cells were resuspended in either DMEM/FCS or defined 
serum-free medium (MEM) and used for experiments. Dissociated rat CNS 
glial cells were prepared starting from optic nerves of 6-7 days old Lewis 
rats as described in Section 6.1.1, supra and plated into poly-D-lysine 
(PLYS) coated wells (100 mm.sup.2, 100 .mu.l medium) at a density of 
20,000 cells per well. The culture medium was an enriched L15 medium with 
5% rat serum, penicillin and streptomycin. C6, 3T3 and B16 cells were 
added to 2 day old cultures at a concentration of 30,000 cells per well, 
incubated for two hours and fixed with warm 4% formalin in phosphate 
buffer. Inhibitory oligodendrocytes were identified by double labelling 
using the specific antibodies O.sub.1 and O.sub.4 (see Section 6.1.3, 
supra). 
9.1.2. Preparation of Nerve Explants for Infiltration Assay 
Optic nerve and sciatic nerve explants were prepared as described (Schwab 
and Thoenen, 1985, J. Neurosci. 5: 2415-2423). Briefly, the nerves were 
rapidly dissected from about 8 week old male rats, cleaned from the 
meninges, 3 times frozen and thawed using liquid nitrogen, and placed 
under a teflon ring (diameter 13 mm, thickness 1 mm) sealed to a culture 
dish with silicon grease. Two chambers connected only by the explants were 
in this way obtained. 300,000 C6, 3T3 or B16 cells were plated in the 
inner chamber in DMEM/FCS and incubated for 5 to 20 days. The medium was 
changed every other day. Cultures were fixed overnight with 4% formalin. 
The nerve explants were mounted with Tissue-Tek, 10 to 15 .mu.m sections 
were cut in a cryostate and collected on gelatine coated cover slips. 
After drying at room temperature overnight, the sections were stained in 
0.75% cresyl violet, and evaluated. The infiltrated cells were counted for 
each 0.1 mm of the explants, starting from the tip where cells were added. 
Due to the 15 day incubation, the explants were often different in 
diameter. Therefore, only the central part of the nerves (0.25 mm) were 
considered, since only this part of the explants presented a good 
histological quality. Inhibition experiments were performed with nerve 
explants previously injected from both sides with 2 .mu.l of 3 mM 
cbz-tyr-tyr or cbz-ala-phe solutions. 
9.1.3. CNS Frozen Sections and Myelin as Substrates 
Adult rat cerebellum frozen sections were prepared and dried on glass 
coverslips. 70,000 C6, 3T3, or B16 cells in 100 .mu.l were added to each 
well containing slices previously rinsed with cold DMEM/FCS. Cultures were 
incubated for 2 days at 37.degree. C. Cultures were then fixed and stained 
with cresyl violet. Three to four cerebellum slices were used per point 
per experiment, with each experiment being repeated at least 2 times. 
Myelin from rat spinal cord (CNS) or sciatic nerve (PNS) purified on a 
discontinuous sucrose gradient as described in Section 6.1.5., was dried 
overnight onto PLYS coated wells (20 .mu.g protein/well of 100 mm 
surface). Unbound membranes were removed by three washes with Ca.sup.2+ 
/Mg.sup.2+ -free Hank's solution. Myelin coated wells were immediately 
used in substrate testing assays by the addition of 9,000 cells (C6, 3T3, 
or B16) per cm.sup.2. Alternatively, we used extracted CNS myelin protein, 
or SDS-PAGE purified 35 and 250 kD inhibitory proteins reconstituted in 
liposomes (see Section 7.1.5, supra). Experiments were scored at different 
time points using a phase contrast microscope equipped with a photocamera. 
Quantifications were done using a surface integration program; three 
arbitrary fields were photographed for each well at a magnitude of 
80.times., at least 25 cells per picture were measured. Each point 
represents the mean of at least 3 wells.+-.SEM. Results are expressed as 
.mu..sup.2 of projected cell surface, or as degree, which was calculated 
by subtracting from the projected surface value of a spreading cell, the 
surface value of a completely spheric cell. 
9.1.4. C6 Plasma Membranes and Conditioned Medium Preparation 
C6 cells grown to 80% confluency were washed twice with Hank's medium, and 
harvested in 20 ml 8.5% sucrose, 50 mM NaCl, 10 mM Tris buffer, pH 7.4, 
using a rubber policeman. After mechanical homogenization through a series 
of needles of decreasing size, a low purity plasma membrane fraction was 
obtained by centrifugation (5 minutes at 3000.times. g, 10 minutes at 
8000.times. g, and then 2 hours at 100,000.times. g). A higher purity 
fraction was isolated by loading the material on a discontinuous sucrose 
gradient, containing 50 mM NaCl, 10 mM Tris, pH 7.4 (Quigley, 1976, J. 
Cell Biol. 71: 472-486). 20-40% sucrose interphase (C6 plasma membranes 
fraction) and 40-60% sucrose interphase (C6 mitochondrial fraction) were 
collected, washed in Hank's medium and resuspended in MEM. 
Conditioned media were obtained by cultivating 80% confluent C6 cell 
cultures for 1 day in MEM. The medium was then collected and centrifuged 
for 10 minutes at 3000.times. g. In some experiments the conditioned 
medium was concentrated 10 times using Centricon Tubes. 
9.1.5. Treatment of CNS Myelin with C6 Plasma Membranes 
CNS myelin coated PLYS wells were prepared as described in the previous 
section, but instead of being immediately tested as substrate, they were 
first incubated with 50 .mu.l of C6 plasma membranes (containing 0.8 mg 
protein/ml MEM) at 37.degree. for 30 minutes. Dishes were then rinsed 
twice with Hank's medium and immediately used as substrates for 3T3 cells. 
In some experiments, protease blockers were added to the membranes using 
10 times concentrated solutions. 
9.2. Results 
9.2.1. C6 Glibobastomas but not 3T3 Fibroblasts or B16 Melanomas Infiltrate 
Optic Nerve and CNS White Matter in vitro 
Frozen optic nerve and sciatic nerve explants were placed under a teflon 
ring and sealed with silicon grease (Schwab and Thoenen, 1985, J. 
Neurosci. 5: 2415-2423). C6 or 3T3 cells were plated into the ring, in 
contact with one end of the nerve explants. Culture medium was exchanged 
every other day, and after 5 to 20 days of incubation the nerves were 
fixed, and sectioned with a cryotome. Infiltrated cells were recognized by 
cresyl violet staining. PNS explants supported diffuse infiltration of 
both C6 and 3T3 cells (FIGS. 19C,D). C6 cells were present in the explants 
at higher density. In the optic nerve explants, a different picture 
emerged (FIGS. 19A,B); 3T3 cells did not infiltrate the nerves, with the 
exception of very few cells which migrated along blood vessels (FIG. 19B, 
arrow). On the other hand, C6 cells infiltrated deep into the optic nerves 
with a diffuse pattern, reaching a maximum distance of about 3 mm from the 
entry point in 14 days (migration rate: about 0.2 mm/day). 
As an alternative model, adult rat cerebellum frozen sections were used as 
a culture substrate for C6, B16 or 3T3 cells. The highly metastatic B16 
melanoma cells were found to clearly discriminate between the substrate 
qualities of the grey and white matter with regard to cell attachment, 
spreading and migration. In fact, B16 cells exclusively attached and 
spread on grey matter regions and, even if plated at high cell densities, 
they did not attach on or migrate into white matter areas of the sections 
(FIGS. 20E,F). The same picture emerged for 3T3 cells, which formed dense 
monolayers on grey matter, but not on white matter (FIGS. 20C,D). In 
contrast to B16 and 3T3 cells, C6 cells were found frequently on white 
matter as well as on grey matter (FIGS. 20A,B). In some cases we found 
that C6 cells were more dense on the white matter than on the molecular 
layer of the grey matter, where they often formed little aggregates which 
spread with difficulty. 
9.2.2. Glioblastoma Cell Spreading is not Inhibited by CNS Myelin 
The spreading behavior of C6 glioblastomas on CNS myelin adsorbed to PLYS 
coated wells was compared to that of B16 melanomas and 3T3 fibroblasts. 
B16 melanoma reaction to a CNS myelin substrate strongly resembled that of 
3T3 fibroblasts: 3T3 or B16 cells spreading on CNS myelin was strongly 
impaired, whereas C6 cell spreading was slightly reduced at the beginning 
(90 minutes), but no further appreciable differences were detected at 
later time points (FIGS. 21A-C). The differences between cells on CNS 
myelin or on PLYS also persisted with prolonged incubation times (up to 1 
day). 
C6 cells were confronted with the SDS-PAGE purified inhibitors (35 kD and 
250 kD) reconstituted in liposomes, and also with living, cultured 
oligodendrocytes. Again, 35 kD and 250 kD liposomes strongly inhibited 3T3 
cell spreading, but they did not impair C6 cell spreading; C6 cells 
adhered and rapidly assumed the well spread characteristic "fried egg" 
appearance also on these reconstituted CNS myelin fractions. 
9.2.3. Specific Blockers of Metalloproteases Inhibit C6 Cell Spreading on 
CNS Myelin 
The involvement of proteases in C6 behavior was investigated by determining 
the effect of inhibitors of proteases on C6 cell spreading on either CNS 
myelin or PLYS. Cys-, Ser- and Asp-protease blockers at the adequate 
concentrations had no discernible effect on C6 spreading on CNS myelin 
(Table X). 
TABLE X 
______________________________________ 
EFFECT OF DIFFERENT PROTEASE INHIBITORS ON C6 
CELL SPREADING ON PLYS OR CNS MYELIN* 
Spreading on: 
Inhi- 
PLYS CNS bition 
Protease 
Protease (% of control 
on CNS 
Class Inhibitor on PLYS) (%) 
______________________________________ 
none, 100 95 5 
control 
serine 
6-amino-capronate 
3.0 mM 93 100 0 
hirudine 1.0 mM nq nq 0 
PMSF 4.0 mM 100 94 6 
trasylol 200.0 U/ml 98 93 5 
cysteine 
leupeptine 0.3 mM 91 83 8 
aspartic 
pepstatine 0.3 mM 98 95 3 
metallo 
1,10-phenanthroline 
0.3 mM 97 30 67 
bestatine 0.1 mM nq 104 0 
phosphoramidon 
0.3 mM nq 91 9 
TIMP 10.0 .mu.g/ml 
102 93 9 
cm-phe--leu 0.5 mM 95 92 3 
cbz--gly--gly--NH.sub.2 
1.0 mM nq 99 1 
cbz--gly--phe--NH.sub.2 
1.0 mM 100 45 55 
cbz--ala--phe 0.3 mM 98 90 8 
cbz--tyr--tyr 0.3 mM 101 56 45 
general 
2-macroglobulin 
3.0 .mu.M 
70 52 18 
cocktail - nq nq 0 
cocktail + nq nq ++ 
______________________________________ 
*Cells were plated on PLYS or CNS myelin coated culture dishes. Spreading 
was determined after 150 minutes as described supra in Materials and 
Methods. Inhibition values were calculated by subtracting spreading value 
on CNS myelin from the values on PLYS. 
PMSF: Phenyl methyl sulfonyl fluoride. 
TIMP: Tissue inhibitor of metalloproteases. 
Cocktail -: trasylol, 200 U/ml; leuptine, 0.3 mM; pepstatine, 0.3 mM. 
Cocktail +: same as cocktail -, but with 0.3 mM 1,10phenanthroline. 
nq: not quantified, only qualitative 
The specific metalloprotease blocker 1,10-phenanthroline on the other hand, 
resulted in a strong inhibition of C6 spreading specifically on CNS 
myelin: 1,10-phenanthroline inhibited C6 spreading on myelin up to 67% 
after 2 hours in culture (Table X). None of the blockers tested showed a 
significant effect on C6 cell spreading on PLYS. 1,10-phenanthroline is a 
general metalloprotease inhibitor due to its property of metal ion 
chelation. However, inhibition by this substance is not sufficient to 
define a proteolytic activity, since other metallodependent enzymes are 
also inhibited. Many other inhibitors of metalloproteases have been found, 
but they usually turned out not to be as general as 1,10-phenanthroline. 
Phosphoramidon (Komiyama, et al., 1975, Biochem. Biophys. Res. Comm. 65: 
352-357), bestatine (Umezawa, et al., 1976, J. Antibiot. 29: 857-859) and 
the tissue inhibitor of metalloprotease (TIMP; Cawston, et al., 1987, 
Biochem. J. 195: 159-165) did not impair C6 cell spreading (Table X). 
TIMP also does not inhibit a brain membrane associated metalloprotease 
degrading enkephaline. Carboxymethyl-phe-leu (Fournie-Zaluski, M. C. et 
al., 1983, J. Med. Chem. 26: 60-65), a modified peptide with high affinity 
for enkephalinase (Almenoff, J. and M. Orlowski, 1983, Biochemistry 22: 
590-599), did not inhibit C6 cell spreading (Table X). On the other hand, 
we found that the dipeptides cbz-gly-phe-NH.sub.2 and cbz-tyr-tyr lead to 
55% inhibition of C6 cell spreading on CNS myelin, but not on PLYS, PNS 
myelin or glass. These peptides are substrate peptides with 
metalloprotease specificity (Almenoff and Orlowski, supra; Baxter, et al., 
1983, Proc. Natl. Acad. Sci. U.S.A. 80: 4174-4178; Couch and Strittmatter, 
1983, Cell 32: 257-265; Chen and Chen, 1987, Cell 48: 193-203; Lelkes and 
Pollard, 1987, J. Biol. Chem. 262: 15496-14505). 
In order to exclude a possible general enhancement of C6 cell spreading on 
nonpermissive substrates, we tested metalloprotease-dependent C6 cell 
spreading on two other substrates in addition to PLYS and CNS myelin (FIG. 
22): PNS myelin and glass. PNS myelin was chosen as a control for the 
general properties of a myelin membrane fraction (e.g., high content of 
lipids), and glass was chosen because of its well known bad substrate 
qualities. Half maximal inhibition of spreading on CNS myelin was obtained 
with 200 .mu.M 1,10-phenanthroline. On PLYS, glass, and PNS myelin (FIG. 
22), 1,10-phenanthroline did not impair C6 cell spreading at 
concentrations up to 0.5 mM (FIG. 22). 
Absorption of CNS myelin with a monoclonal antibody (IN-1) raised against 
CNS myelin inhibitory components (see Section 8.1.3., supra) largely 
reversed 1,10-phenanthroline dependent inhibition of C6 cell spreading on 
CNS myelin liposomes (Table XI). IN-1 also almost completely neutralized 
the inhibitory substrate property of CNS myelin protein liposomes for 3T3 
cells (Table XI). 
TABLE XI 
______________________________________ 
INHIBITION OF C6 CELL SPREADING BY 
1,10-PHENANTHROLINE ON CNS MYELIN 
IS NEUTRALIZED BY ANTIBODY IN-1* 
% inhibition 
1,10-Phenan- 
Spreading value on: 
on CNS 
Cells 
Antibody throline CNS lipos. 
PLYS lipos. 
______________________________________ 
3T3 -- 0 1.11 2.00 45 
3T3 IN-1 0 2.03 2.26 10 
3T3 mouse IgM 0 1.16 2.18 47 
C6 -- 0 2.48 2.52 2 
C6 -- 0.3 mM 1.35 2.49 46 
C6 IN-1 0 2.46 2.48 1 
C6 IN-1 0.3 mM 2.25 2.54 11 
C6 mouse IgM 0 2.36 2.42 2 
C6 mouse IgM 0.3 mM 1.41 2.39 41 
______________________________________ 
*CNS myelin protein liposomes were used as substrates, and were 
preadsorbed with monoclonal antibody IN1 against the myelin inhibitory 
substrate constituents (see Section 8.1.4), or with mouse IgM. Spreading 
was calculated after 150 minutes and is expressed as .mu.m.sup.2 10.sup.3 
% Inhibition relates to spreading values on PLYS. 
These results indicate that the metalloprotease(s) plays an important role 
for overcoming of CNS myelin inhibitory substrates by neutralization of 
IN-1 inhibitory properties. 
9.2.4. A C6 Plasma Membrane Associated Activity Neutralizes the Inhibitory 
Substrate Property of CNS Myelin 
CNS myelin-coated culture wells were incubated with C6 conditioned medium 
or C6 plasma membranes, and subsequently tested for their inhibitory 
substrate property with spreading of 3T3 cells. We found that C6 plasma 
membranes contained an activity which strongly reduced CNS myelin 
inhibitory activity (FIGS. 23A-D, Table XII). The same treatment also 
decreased the inhibitory effect of CNS myelin protein liposomes or 
SDS-PAGE-purified, reconstituted 35 kD and 250 kD inhibitory components. 
The decrease in CNS myelin inhibitory activity for 3T3 cell adhesion and 
spreading was quantitified by measuring spreading values and DNA synthesis 
(Table XII). 
TABLE XII 
______________________________________ 
C6 PLASMA MEMBRANES REDUCE CNS MYELIN 
INHIBITORY SUBSTRATE PROPERTY FOR 3T3 CELLS* 
3T3 Cell .sup.3 H-Thymidine 
Spreading 
Incorporation 
Substrates (%) (%) 
______________________________________ 
PLYS 100 100 
CNS myelin 15 30 
CNS myelin, C6 PM 52 83 
CNS myelin, C6 PM, phen. treated 
17 50 
CNS myelin, C6 PM, EDTA treated 
13 nd 
______________________________________ 
*3T3 cells were plated on PLYS or CNS myelin. Spreading was assessed afte 
150 minutes CNS myelin was preincubated with a C6 cell plasma membrane 
fraction (C6 PM) in the absence or presence of metalloprotease inhibitors 
as indicated. .sup.3 Hthymidine was added when 3T3 cells were plated, and 
incorporation was determined after 20 hours. 
nd: not determined 
1,10-phenanthroline, EDTA, and the dipeptide cbz-gly-phe-NH.sub.2 
completely blocked the C6 plasma membrane effect. Trasylol, leupeptine and 
pepstatine did not inhibit this effect. C6 conditioned medium used as 
such, or 10-times concentrated, did not contain any degradative activity 
able to neturalize CNS myelin inhibitory substrate properties. 
9.2.5. Inhibitors of Metalloproteases Impair C6 Cell Spreading on CNS White 
Matter and C6 Infiltration of CNS Explants 
In order to investigate the relevance of the C6 plasma membrane 
metalloprotease activity not only for C6 cell attachment and spreading, 
but also for C6 cell migration and infiltration, C6 cells were plated on 
cerebellar frozen sections or added to optic nerve explants in the 
presence of two metalloprotease inhibitors (1,10-phenanthroline and 
cbz-tyr-tyr). Parallel cultures contained inhibitors for the three other 
classes of proteases (leupeptine, pepstatine or trasylol), or a control 
dipeptide (cbz-ala-phe). 
The presence of 1,10-phenanthroline at different concentrations (50, 100, 
200 and 300 .mu.M), or the dipeptide cbz-tyr-tyr (100 .mu.M) dramatically 
changed the distribution and behavior of C6 cells on the white matter 
areas when cerebellar frozen sections were used as culture substrates 
(FIGS. 23A-D). C6 cells also adhered in large numbers and spread 
extensively on the grey matter (FIGS. 23A-D). 
Rat optic nerves were injected with 4 .mu.l of 3 mM solutions of either 
cbz-ala-phe or cbz-tyr-tyr. Cells were incubated with medium containing 
0.5 mM peptide. In the outer chamber, where no cells were present, the 
peptide concentration was 1 mM. After 14 days, the immigration of C6 cells 
into the explants differed greatly (FIGS. 24A-B). Cbz-ala-phe-injected 
nerves contained more cells, and C6 cell infiltration was not affected, as 
compared to explants injected with culture medium only. On the other hand, 
cbz-tyr-tyr inhibited C6 cell infiltration in all the 8 nerves examined (2 
experiments). C6 cells were found mainly at the cut end of these nerve 
explants, and deep infiltration, which occurred massively in control 
explants, was strongly reduced by cbz-tyr-tyr. 
9.3. Discussion 
The present results demonstrate that C6 glioblastoma cells, in contrast to 
neurons, fibroblasts and B16 melanoma cells, were not impaired in their 
migration into optic nerve explants or in attachment and spreading on CNS 
white matter, isolated CNS myelin, or living oligodendrocytes. The fact 
that the behavior of C6 cells differed characteristically from that of 
several cell types in all the assay systems studied suggests common 
underlying cell biological mechanisms, both for C6 spreading on an 
inhibitory substrate as well as for C6 mobility in an environment (optic 
nerve) which does not allow fibroblasts, Schwann cell or melanoma cell 
migration nor does it allow ingrowth of regenerating nerve fibers. This 
behavior of C6 cells was not due to "insensitivity" to the inhibitory 
components, since C6 cell motility was drastically inhibited on CNS myelin 
or white matter in the presence of specific protease blockers, and this 
effect was reversed by selective neutralization of myelin-associated 
inhibitory proteins with a monoclonal antibody (IN-1). 
Inactivation of myelin-associated inhibitory constituents occurred by 
living C6 cells as well as by C6 plasma membranes. Our experiments with a 
number of protease blockers with different known specificities showed that 
this C6 associated activity belongs to the metalloenzyme family. The close 
parallelism observed between prevention of C6 cell spreading on CNS myelin 
and prevention of inactivation of myelin-associated inhibitory proteins 
strongly suggests that modification of the inhibitory substrate components 
by a metalloprotease could be the mechanism which enables C6 cells to 
spread on myelin, on white matter, and to infiltrate optic nerve explants. 
Metalloproteases form an increasingly numerous group, the members of which 
differ in their sensitivity to various blockers. The most general blocker 
is 1,10-phenanthroline which impaired C6 cell spreading on CNS myelin up 
to 67%, whereas most inhibitors of the other classes of proteases had no 
detectable effects. In the early (90 minutes) but not the later (300 
minutes) phases of C6 cell spreading on myelin, an effect of trypsin-like 
serine-protease inhibitors was also observed. The effect of 
1,10-phenanthroline was dose-dependent, with an IC.sub.50 of 200 .mu.M. 
This effect was specific for CNS myelin as a substrate, since normal, 
rapid spreading of C6 cells was observed on other substrates such as CNS 
grey matter, PNS myelin, glass or PLYS in the presence of 
1,10-phenanthroline. Other known metalloprotease blockers like bestatine 
(inhibitor of aminopeptidases; Umezawa, et al., 1976, J. Antibiot. 29: 
857-859), phosphoramidone (inhibitor of thermolysin-like metalloproteases; 
Komiyama, et al., 1975, Biochem. Biophys. Res. Commun. 65: 352-357) and 
TIMP (inhibitor of ECM degrading metalloproteases; Cawston, et al., 1981, 
195: 159-165) did not lead to inhibition of C6 cell spreading on CNS 
myelin. Since metalloproteases generally hydrolyze peptide bonds followed 
by large aliphatic or neutral aromatic amino acids, we tested the effect 
of dipeptide substrate analogues containing such residues. 
Cbz-gly-phe-NH.sub.2 (1 mM) or cbz-tyr-tyr (0.3 mM) inhibited C6 cell 
spreading specifically on CNS myelin. Cbz-gly-phe-NH.sub.2 was found to 
inhibit other 1,10-phenanthroline sensitive enzyme activities with 
relative high specificity (Almenoff and Orlowski, 1983, Biochemistry 22: 
590-599; Baxter, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 
4174-4178; Couch and Strittmatter, 1983, Cell 32: 257-265; Chen, J. M. and 
Chen, W. T., 1987, Cell 48: 193-203; Lelkes and Pollard, 1987, J. Biol. 
Chem. 262: 15496-14505). 
Inactivity of C6-conditioned medium and cell fractionation experiments 
demonstrated that the myelin-directed proteolytic activity is associated 
with C6 plasma membranes. The isolation and characterization of a plasma 
membrane-bound metalloprotease (endopeptidase 24.11, enkephalinase), which 
is also blocked by 1,10-phenanthroline but not by TIMP, was reported by 
Almenoff and Orlowski (1983, supra). However, the metalloprotease 
described herein is probably not an enkephalinase, since 
carboxymethyl-phe-leu, a peptide with high affinity for enkephalinase 
(Fournie-Zaluski, et al., 1983, J. Med. Chem. 26: 60-65), did not affect 
C6 spreading on myelin. A metalloprotease expressed by Rous sarcoma virus 
transformed chick embryo fibroblasts and localized at adhesion sites and 
on "invadopodia" was described by Chen, and Chen, 1987, supra. This enzyme 
is also inhibited by 1,10-phenanthroline and cbz-gly-phe-NH.sub.2, but not 
by phosphoramidon, as is the metalloprotease described here. However, 
unlike the enzyme of Chen and Chen, we could not detect any fibronectin 
degradative activity on C6 cells. 
The highly metastatic B16 mouse melanoma cells were tested in all the 
assays used with C6 cells. Interestingly, B16 cells did not migrate into 
optic nerve explants, but responded to the myelin-associated inhibitors in 
a way very similar to 3T3 cells or neurons. In line with this in vitro 
behavior, B16 cells, upon intraventricular injection, form mainly 
meningiomas or intraventricular tumors without significant infiltration of 
the brain parenchyma. Thus, the mechanisms providing metastatic behavior 
to B16 cells in the periphery are different from those conferring high 
mobility to C6 cells in the CNS tissue. 
Inhibition of C6-associated metalloprotease not only inhibited C6 spreading 
on CNS myelin, but also abolished C6 cell attachment, spreading, and 
migration on CNS white matter, and the dipeptide, cbz-tyr-tyr strongly 
impaired the migration of C6 cells into optic nerve explants. This 
metalloprotease activity(ies) may, therefore, be crucially involved in the 
infiltrative behavior of C6 glioblastoma cells in CNS tissue, also in 
vivo. 
10. LONG DISTANCE TRACT REGENERATION IN THE LESIONED SPINAL CORD OF RATS BY 
A MONOCLONAL ANTIBODY AGAINST MYELIN-ASSOCIATED NEURITE GROWTH INHIBITORS 
The monoclonal antibody IN-1, which neutralizes the inhibitory substrate 
effect of the 35 kD and 250 kD myelin-associated proteins and of CNS 
tissue explants (Caroni and Schwab, 1988, Neuron 1: 85-96), was applied to 
young rats intracerebrally by implanting antibody producing tumors into 
the neocortex. Complete transections of the cortico-spinal component of 
the pyramidal tract (CST) at 2-4 weeks of age was followed by massive 
sprouting around the lesion, and, in IN-1 treated rats, by elongation of 
fine axons and fascicles up to 8-11 mm distal to the lesion within 2 
weeks. In control rats the maximal distance of observed elongation rarely 
exceeded 1 mm. These results demonstrate the induced regeneration capacity 
of a major motor CNS tract within differentiated CNS tissue, and point to 
the clinical importance of CNS neurite growth inhibitors and their 
antagonists. 
10.1. Materials and Methods 
10.1.1. Pre-Operative Preparation of Animals Including Implantation of 
Hybridoma Cells 
Young Lewis rats (P2-11) were injected unilaterally under ether anesthesia 
into the dorsal frontal cortex with 1 Mio. hybridoma cells in 1 or 2 
.mu.l. Control rats were injected with the same number of cells of a 
hybridoma line producing antibodies against horseradish peroxidase (HRP). 
Non-injected controls were also used. Hybridoma cells: IN-1 secreting 
cells were obtained by fusion of P3U myeloma cells with spleen cells of a 
BALB/c mouse immunized against the PAGE-purified 250 kD inhibitory protein 
fraction from rat spinal cord myelin as described by Caroni and Schwab 
(1988, Neuron 1: 85-96); anti-HRP secreting cells were obtained by Dr. P. 
Streit, Zurich, according to the protocol of Semenenko et al. (1985 
Histochem. 83: 405-408) using the same myeloma line (P3U) as for IN-1. In 
all hybridoma-injected rats, tumors formed within a few days as solid, 
well delineated tumors often spanning the entire thickness of the 
neocortex and contacting the lateral ventricle (FIG. 26A-B). Cyclosporin A 
injections (15 .mu.g/g body weight, 2 injections at 3 day intervals) 
helped to prevent tumor resorption which otherwise occurred after 2-3 
weeks. Massive production of antibodies could be detected by staining 
brain sections with anti-mouse Ig-FITC (FITC-conjugated immunoglobulin) 
(FIG. 26B), and by the presence of In-1 antibodies in the serum (data not 
shown). 
10.1.2. Procedure for Performing Spinal Cord Lesion 
Spinal cord lesions were placed at 2-4 weeks of age (Table III, infra) at 
the thoracic level T.sub.5-7 by slightly separating two vertebrae and 
transecting the dorsal two thirds of the spinal cord with iridectomy 
scissors. The lesion completely transsected the CSTs of both sides 
including the lateral projections into the dorsal gray matter, and also 
the central canal. Ventral and lateral white matter remained undisturbed, 
allowing the rats a seemingly normal behavior. Lesions were done at 15-29 
days of age, i.e. 5-20 days after termination of axon growth in the CST 
(Table XIII). A U-shaped stainless steel wire was then implanted into the 
lesion site in order to assure complete transection of both CSTs and to 
mark the lesion site. (The wire was removed prior to embedding the fixed 
spinal cords for sectioning). 
TABLE XIII 
______________________________________ 
REGENERATION OF CORTICO-SPINAL TRACT AXONS 
AFTER MID-THORACIC LESIONS IN CONTROL AND 
ANTIBODY IN-1 TREATED RATS 
Tumor- Day of Survival 
Max. distance of regenerated 
type lesion time CST axons caudal to lesion 
______________________________________ 
none P 14 19 d. 0.1 
0.2 
0.2 
0.5 
none P 22 14 d. 0.4 
0.2 
none P 22 11 d. 0.7 
0.6 
.alpha.HRP 
P 15 14 d. 0.4 
1.0 
1.8 
2.6 
.alpha.HRP 
P 18 16 d. 0.1 
0.2 
0.2 
0.3 
0.4 
0.5 
0.8 
IN-1 P 14 16 d. 2 
&gt;8* 
11 
IN-1 P 15 15 d. 4 
4.5 
&gt;5* 
&gt;5 
&gt;5 
IN-1 P 18 18 d. 2.5 
&gt;3 
IN-1 P 19 14 d. 7.7 
7.8 
IN-1 P 28 14 d. &gt;4 
&gt;4 
IN-1 P 29 27 d. &gt;2.5 
______________________________________ 
Methods as described in FIG. 27. Only rats with regenerative CST sprouts 
caudal to the lesion were included in this analysis. Distances of 
regenerating fibers are measured from the caudal edge of the lesion 
caverns. 
*Minimal distance as regenerating fibers reach the caudal end of the 
tissue block. 
10.1.3. Post-Lesion Evaluation 
After survival times of 14-28 days (Table XIII), the frontal and parietal 
cortex contralateral to the tumor was injected with a 5% solution of 
WGA-HRP (1 .mu.l). Twenty-four hours later, rats were perfused through the 
heart with 1.25% glutaraldehyde and 1% formaldehyde in 0.1M phosphate 
buffer for 10 minutes. The dissected spinal cords (10-15 mm) were 
postfixed in the same fixative for 1 hour, extensively washed, and embeded 
for cryostat sectioning. Complete longitudinal section series were mounted 
on gelatin-coated slides, and reacted for HRP using TMB as a substrate 
(Mesulam, 1978, J. Histochem. & Cytochem. 26: 106-117). Sections were 
viewed under dark-field illumination in polarized light. Only rats with 
complete bilateral CST lesions and with sprouts appearing on the caudal 
side of the lesion were evaluated. 
10.2. Results: Regeneration of Corticospinal Tract (CST) Fibers Over Long 
Distances in Rats Bearing IN-1 Secreting Tumors 
Two weeks after the lesions, at or beyond 1 month of age, the CST was 
labeled by anterograde transport of WGA-HRP from the frontal and parietal 
cortex. The histological examination of the lesion site in transverse and 
longitudinal sections showed a very similar picture in all animals: 
usually several small caverns were present and communicated with the 
central canal, a feature which probably greatly enhanced the local access 
and penetration of the antibodies carried down by the cerebrospinal fluid. 
The tissue was locally altered, but no dense glial scars were present. 
Labeled CST fibers approached the lesion as a dense and compact bundle 
from which massive sprouting occurred 0.5-1 mm proximal to the lesion. In 
most animals, controls or IN-1-injected, fiber plexus and bundles were 
seen in and across the lesion area, most often circumventing the lesion 
caverns ventrally or laterally, but rarely also growing through tissue 
bridges that had reformed in the wire tract. Fibers leaving the lesion 
site and travelling in a caudal direction could frequently be observed. In 
animals without tumors and in rats with anti-HRP-producing tumors, the 
travelling distances measured on longitudinal sections from the distal 
edge of the lesion were in most instances below 1 mm (Table XIII, FIGS. 
27,28A-G). Even relatively thick fascicles seemed to end abruptly. Very 
much in contrast, animals bearing IN-1 secreting tumors consistently 
showed labelled fascicles and fibers at much longer distances caudal to 
the lesion (FIGS. 27,28A-G). 2.5-5 mm were measured in most animals, 8 and 
11 mm were seen in 2 rats (Table XIII). Anatomically, these long distance 
regenerating CST fibers were most often found close to or in the original 
CST location, with some fibers also in the gray matter and a few fibers in 
more dorsal regions corresponding to the sensory tracts. 
10.3. Discussion 
In the rat, the CST is known to grow down the spinal cord during the first 
10 postnatal days, the last axons being added at P9-P10 (Joosten et al., 
1987, Dev. Brain Res. 36: 121-139; Schreyer and Jones, 1988 Dev. Brain 
Res. 38: 103-119). Lesions of the tract up to P4-P5 lead to a 
circumvention of the lesion site and to long-distance, often ectopic 
growth of CST fibers (Schreyer and Jones, 1983, Neurosci. 9: 31-40; 
Bernstein and Stelzner, 1983, J. Comp. Neurol. 221: 382-400). No 
regeneration in the CST has been seen after P6. A very similar lesion 
response has been observed in hamster and cat (Kalil and Reh, 1982, J. 
Comp. Neurol. 211: 265-275; Tolbert and Der, 1987, J. Comp. Neurol. 260: 
299-311). For the cat it was demonstrated that these fibers are mostly 
late-arriving, newly-growing, rather than regenerating axons (Tolbert and 
Der, 1987, J. Comp. Neurol. 211: 265-275). The present results demonstrate 
that at least a small proportion of CST neurites at 2-3 weeks of age can 
be induced to regenerate and elongate over long distances inside the 
spinal cord. The maximal speed of elongation is in the range of 0.5-1 
mm/day. 
Differentiated CNS tissue of mammals is a nonpermissive substrate for 
neurite growth beyond a sprouting distance of between 0.2-1 mm (Cajal, 
1959, in "Degeneration and Regeneration of the Nervous System," ed. 
Hafner, New York, p. 1928; David, 1981, Science 214: 931-933; and 
Vidal-Sanz et al., 1987, J. Neurosci. 7: 2894-2909). This property is 
expressed (far more by CNS white matter than CNS gray matter, as shown by 
culture experiments and by transplantation studies (Schwab and Thoenen, 
1985, J. Neurosci. 5: 2415-2423; Carbonetto et al., 1987, J. Neurosci. 7: 
610-620; Savio and Schwab, 1989, in press). Transplantations of fetal 
adrenergic or serotoninergic neurons of defined fetal ages into adult 
spinal cords or hippocampus represent up to now the only other experiments 
where elongation of axons in adult CNS tissue was observed at an 
anatomical level (Nornes et al., 1983 Cell Tissues Res. 230: 15-35; Foster 
et al., 1985 Exp. Brain Res. 60: 427-444 and Bjorklund et al., 1979 Brain 
Res. 170: 409-426). These elongating axons were almost exclusively 
localized to gray matter areas. 
Two oligodendrocyte- and myelin-associated membrane proteins, NI-35 (35 kD) 
and NI-250 (250 kD), with potent inhibitory effects on neurite growth, 
were identified by in vitro and biochemical studies (Schwab and Caroni, 
1988 J. Neurosci. 8: 2381-2393; Caroni and Schwab, 1988, J. Cell. Biol. 
106: 1281-1288). Monoclonal antibody IN-1, which neutralizes the activity 
of these constituents in various in vitro systems including adult rat 
optic nerve explants (Caroni and Schwab, 1988, Neuron 1: 85-96), is shown 
here to lead to true regeneration of cortico-spinal axons in young rats 
over distances of up to 5-11 mm distal to a spinal cord lesion within 2 
weeks. The continuous supply of high levels of antibodies via the 
cerebrospinal fluid by an antibody-secreting tumor in the cortex, and the 
local conditions of the lesion probably helped the penetration of the 
antibodies into the tissue. The absence of axon elongation distal to the 
lesion in spite of massive sprouting around the lesion site in animals 
bearing control antibody tumors confirms the specificity of the effect 
observed. These results clearly demonstrate the ability of antibodies 
directed toward the myelin-associated neurite growth inhibitor protein to 
induce neuron fiber regeneration over long distances, as well as the 
crucial role of the myelin-associated neurite growth inhibitors for the 
absence of regeneration of lesioned CNS fiber tracts observed under normal 
conditions. 
11. DEPOSIT OF MICROORGANISMS 
The following hybridomas, producing the indicated monoclonal antibodies, 
have been deposited on Oct. 28, 1988 with the European Collection of 
Animal Cell Cultures (ECACC), PHLS Centre for Applied Microbiology and 
Research, Porton Down, Salisbury, Wiltshire, United Kingdom, and have been 
assigned the listed accession numbers. 
______________________________________ 
Hybridoma Antibody Accession Number 
______________________________________ 
Cell line IN-1 
IN-1 88102801 
Cell line IN-2 
IN-2 88102802 
______________________________________ 
The present invention is not to be limited in scope by the cell lines 
deposited or the embodiments disclosed in the examples which are intended 
as illustrations of a few aspects of the invention and any embodiments 
which are functionally equivalent are within the scope of this invention. 
Indeed, various modifications of the invention in addition to those shown 
and described herein will become apparent to those skilled in the art and 
are intended to fall within the scope of the appended claims.