EP0493533A1 - Hemmung des zellwachstums mittels keratansulfat, chondroitinsulfate, dermatansulfat und sonstige glykane - Google Patents

Hemmung des zellwachstums mittels keratansulfat, chondroitinsulfate, dermatansulfat und sonstige glykane

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Publication number
EP0493533A1
EP0493533A1 EP90917627A EP90917627A EP0493533A1 EP 0493533 A1 EP0493533 A1 EP 0493533A1 EP 90917627 A EP90917627 A EP 90917627A EP 90917627 A EP90917627 A EP 90917627A EP 0493533 A1 EP0493533 A1 EP 0493533A1
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EP
European Patent Office
Prior art keywords
patient
sulfate
effective amount
molecule
glial cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP90917627A
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English (en)
French (fr)
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EP0493533A4 (en
Inventor
Diane M. Snow
Jerry Silver
Adrian Harel
Dikla Roufa
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Case Western Reserve University
Gliatech Inc
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Case Western Reserve University
Gliatech Inc
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Publication of EP0493533A1 publication Critical patent/EP0493533A1/de
Publication of EP0493533A4 publication Critical patent/EP0493533A4/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds

Definitions

  • the present invention relates to compositions comprising keratan sulfate, chondroitin sulfate, or dermatan sulfate, also heparan sulfate, heparin, or hyaluronic acid (hyaluronate) , or any combination of these molecules—in particular, glycosaminoglycans or proteoglycans—and the uses of such compositions in inhibition of neurite outgrowth and glial cell invasion or migration.
  • compositions comprising an antagonist of inhibition mediated by keratan sulfate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, or hyaluronate such as antibodies to keratan sulfate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin or hyaluronate; enzymes that degrade keratan sulfate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, or hyaluronic acid; lectins specific for keratan sulfate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, or hyaluronic acid, or disaccharide antagonists of the receptors for
  • compositions of the invention are provided.
  • BACKGROUND OF THE INVENTION 2.1. AXONAL GROWTH Axons grow in stereotyped patterns toward their targets during development of the nervous system. During this directed elongation, axonal growth cones undergo multiple interactions with components of the environment such as the extracellular matrix (Carbonetto et al., 1982, Science 216:897-899; Hankin, M. H. and Silver, J., 1986, Mechanisms of axonal guidance: the problem of intersecting fiber systems, in The Cellular Basis of Morphogenesis, (Leon W. Browder, Ed.) Vol. 2:565-599, Plenum Publishing Corp., New York, New York; Mirsky et al., 1986, J.
  • Inhibitory components may take the form of cellular boundaries or barriers along an axon pathway (Silver, J., 1984, J. Comp. Neurol. 223:238-251) and they may act by mechanical as well as chemical means (Silver, J., and Rutishauser, U.
  • Axon inhibition can occur between different classes of neurons (Kapfhammer, J. P., and Raper, J. A., 1987, J. Neurosci. 7(1) :201-212) , in association with glial cells (Silver et al., 1982, J. Comp. Neurol.
  • roof plate located at the dorsal midline of the developing vertebrate spinal cord (His, W. , 1891, I. Verlangertes Mark, 29:1-74; Ramon y Cajal, S., 1911, Histologie du syste e nerveux de l'homme et des vertebres. (francaise rev. et mise a jour par l'êt, Ed.) Vol. 1. A., Maloine, Paris).
  • This region is comprised of primitive glial cells as determined morphologically (His, 1891, supra) , by the use of tritiated thymidine autoradiography (Altman, J., and Bayer, S. A., 1984, in Advances in Anatomy, Embryology and Cell Biology, Vol. 85, pp. 53-83, Springer-Verlag, Heidelberg, Germany) and with the use of antibodies RC1 and RC2 which specifically label embryonic radial glial cells (Edwards et al., 1986, 8C7, Society for Neuroscience Abstract 12:182).
  • the roof plate contains transient channels which are first observed as a single row of extracellular spaces at E9 (Snow, D.
  • the roof plate undergoes a gradual change in morphology between E12.5, when it has a wedge shape, and E15.5 when it becomes a long, thin septum-like structure at the dorsal midline of the spinal cord in rat.
  • a dorsal subpopulation of the early ventral co missural axons as .well as primary afferents from the dorsal root ganglia come in close proximity to the roof plate. Even though both axon systems have potential targets or pathways in the contralateral spinal cord, they do not cross the roof plate to reach them.
  • Figure 1 is a schematic diagram which depicts the relationship of the commissural and sensory axon systems to the roof plate at E13.5 and E15.5 in rat.
  • rat Smith, C. L. , 1983, J. Comp. Neurol. 220:29-43
  • frog Nedlander, R. and Singer, M. , 1982, Exp. Neurol. 75:221-228
  • a population of sensory axons do cross the dorsal spinal cord just below the posterior columns to form the dorsal commissure.
  • PROTEOGLYCANS are molecules found in abundance in connective tissue, which consist of about 50-95% polysaccharide and about 5-50% protein.
  • Glycosaminoglycans are the polysaccharide chains of proteoglycans, and contain repeating units of disaccharides which consist of an aminosugar derivative, either glucosamine or galactosamine. -6-
  • a negatively charged carboxylate or sulfate group is found in at least one of the sugar units of the disaccharide.
  • Common glycosaminoglycans include hyaluronate (HA) , chondroitin sulfate (CS) , keratan sulfate (KS) , dermatan sulfate (DS) , heparan sulfate (HS) , and heparin (HN) .
  • glycosa inoglycan chains of proteoglycans are found covalently attached to a polypeptide backbone called the core protein (Stryer, L. , 1981, Biochemistry, 2d ed. , W. H. Freeman & Co., New York, pp. 200-203).
  • a keratan sulfate proteoglycan has been identified in the rat cerebral cortex (Vitello et al., 1978, Biochim. Biophys. Acta 539:305-314) and in corpora amylacea of human brain (Liu, H. M. et al., 1987, J. Neuroimmunol. 14:49-60).
  • proteoglycans have been shown to exert a wide spectrum of effects on the migratory behavior of a variety of different cell types (Walicke, P.A. , 1988, Exp. Neurol. 102:144-148; Daman et al., 1988, J. Cell Physiol. 135:293-300).
  • Perris and Johansson (1987, J. Cell Biol. 105:2511-2521) have shown that a form of chondroitin sulfate proteoglycan is inhibitory to the migration of neural crest cells in vitro.
  • This proteoglycan in high concentrations, also inhibited the attachment and neurite formation of human neuroblasto a cells on a cholera toxin B/ganglioside GMl-binding substratum (Mugnai et al., 1988, Exp. Cell Res. 175:299- 247).
  • glycosaminoglycans principally heparan sulfate and dermatan sulfate, were identified as mediators of fibroblast (murine 3T3 cell) attachment to fibronectin.
  • the heparan and dermatan GAGs bound to serum fibronectin covalently attached to Sepharose, while other proteoglycans, notably various chondroitin sulfates and under-sulfated heparan sulfate, did not bind to the column (Laterra, et al., 1980, Proc. Natl. Acad. Sci. U.S.A. 77:6662-6666).
  • the present invention relates to the discovery that keratan sulfate (KS) , chondroitin sulfate (CS) , dermatan sulfate (DS) , heparan sulfate (HS) , heparin (HN) and/or hyaluronic acid (HA) can inhibit neurite outgrowth, i.e., axonal growth, and glial cell migration or invasion.
  • neurite outgrowth i.e., axonal growth, and nerve regeneration herein may be referred to as "nerve growth.”
  • the present invention is directed to methods of using keratan sulfate, and molecules and compositions comprising keratan sulfate, to inhibit or prevent neurite outgrowth and/or glial cell migration or invasion, or nerve or glial cell regeneration.
  • the methods to inhibit neurite outgrowth or glial cell migration or invasion may be used therapeutically, where that is desired.
  • Such molecules comprising keratan sulfate include but are not limited to keratan sulfate glycosaminoglycan and keratan sulfate proteoglycan, with keratan sulfate proteoglycan most preferred.
  • the invention is further directed to molecules and compositions comprising chondroitin sulfate, and the therapeutic uses thereof to inhibit or prevent neurite outgrowth or glial cell migration or invasion.
  • Molecules comprising chondroitin sulfate include but are not limited to chondroitin sulfate glycosaminoglycan, or more preferably, chondroitin sulfate proteoglycan.
  • the invention also encompasses molecules and compositions comprising dermatan sulfate and the therapeutic uses thereof to inhibit or prevent neurite outgrowth or glial cell migration or invasion.
  • Molecules of dermatan sulfate include but are not limited to dermatan sulfate glycosaminoglycan, or more preferably dermatan sulfate proteoglycan.
  • inhibitors and antagonists of keratan sulfate, chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin and/or hyaluronic acid, and molecules and compositions containing the same may be used to promote neurite outgrowth or glial cell migration or invasion and can be administered therapeutically.
  • inhibitors and antagonists include but are not limited to antibodies to KS, CS, DS, HS, HN or HA, and derivatives or fragments thereof, enzymes that degrade KS, CS, DS, HS, HN or HA, lectins specific for KS, CS, DS, HS, HN or HA, and disaccharide antagonists of receptors specific for KS, CS, DS, HS, HN or HA.
  • promotion of neurite outgrowth and glial cell migration or invasion occurs by removing the inhibitory influence of molecules comprising KS, CS, DS, HS, HN or HA, thus allowing promotion of neurite outgrowth or glial cell migration or invasion by endogenous or exogenously added molecules.
  • molecules comprising keratan sulfate can be used together with molecules comprising another glycosaminoglycan or the disaccharide unit thereof, preferably chondroitin sulfate, in the methods of the invention.
  • the present invention also provides pharmaceutical compositions comprising effective amounts of the molecules and compositions comprising keratan sulfate and/or chrondroitin sulfate, dermatan sulfate, heparan sulfate, heparin or hyaluronate.
  • immunocytochemical localization data is presented which indicates that keratan sulfate, alone or in combination with other molecules such as chondroitin sulfate, may be in part responsible for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.
  • keratan sulfate/chondroitin sulfate proteoglycan or dermatan sulfate proteoglycan actively inhibits neurite elongation in a concentration dependent manner.
  • dermatan sulfate and keratan sulfate/chondroitin sulfate inhibit invasion or migration of glial cells, including astrocytes, in a concentration dependent manner.
  • ChE cholinesterase
  • DRG dorsal root ganglion
  • DS-PG dermatan sulfate proteoglycan
  • GAG glycosaminoglycan
  • HA hyaluronic acid, hyaluronate -12-
  • KS/CS-PG keratan sulfate/chondroitin sulfate proteoglycan
  • NCAM neural cell adhesion molecule
  • PBS phosphate buffered saline
  • RCS rat chondrosarcoma tumor cell line cartilage chondroitin sulfate proteoglycan
  • TBS/BSA Tris-buffered saline/bovine serum albumin
  • TPA tetragonolobus purpureas agglutinin (lotus tetragonolobus, lotus lectin)
  • FIGURES Figure 1 Schematic diagram of embryonic day 13.5 (E13.5) and E15.5 rat cervical spinal cord. The relationship of the roof plate (RP) to the developing dorsal column (sensory) axons (SA) and the commissural axons (CA) is depicted. The earliest dorsal population of commissural axons originate near the roof plate. The axons elongate dorsolaterally, then travel ventrally near the periphery of the cord to decussate at the floor plate (FP) .
  • RP roof plate
  • SA developing dorsal column
  • CA commissural axons
  • the primary sensory afferents (SA) wait in the dorsal root entry zone in an oval bundle on E13.5, travelling rostrally and caudally for a few segments.
  • the dorsal column axons move medially with development and abut the roof plate by E15.5. Like the commissural axons, the dorsal column axons respect the dorsal midline barrier.
  • FIG. 2 Traverse 1 ⁇ m plastic section of Ell.5 rat cervical spinal cord.
  • the roof plate (RP) cells are beginning to form the wedge shape which will become most apparent on E13.5.
  • the cells are arranged in an arching pattern in comparison to adjacent neuroepithelial cells which are more linear. Extracellular space between the presumptive roof plate glial cells is not yet significant in comparison to the spaces seen betwen the cells of the remainder of the cord (compare with Fig. 3A) ; cc, central canal, (250X) ,
  • B A 10 ⁇ cryostat section of rat cervical spinal cord on day Ell.5 labelled with 1C12 antibody which stains the ventral commissural axons.
  • FIG. 3 The roof plate of E13.5 rat cervical spinal cord.
  • the roof plate (RP) glia extend an apical process to the pial surface, terminating in an endfoot. Interspersed among these glial cells is an extensive network of large extracellular spaces. Together, the cells and spaces form a wedge-shaped region at the dorsal aspect of the spinal cord. Compare the cells of the roof plate with the surrounding region of cells which are closely apposed to one another. The surrounding cells and their processes, some of which are commissural neurons, arch dorsolaterally along the perimeter and then away from the roof plate, (630X) .
  • (B) Transverse frozen section of the same age and cord level as in (A) , labelled with an anti-keratan sulfate (a-KS) monoclonal antibody. Keratan sulfate epitopes are specific to the roof plate (RP) at this stage of development. Note that the labelling pattern coincides directly with the wedge-shaped region of glial cells and interspersed extracellular spaces of the roof plate seen in (A) ; cc, central canal, (63OX) .
  • FIG. 4 Transverse frozen section of E13.5 rat cervical spinal cord labelled with antibody 1C12.
  • the commissural axons (ca) take a stereotypical path away from the roof plate (RP) as they course from the dorsolateral wall of the spinal cord along the periphery to the floor plate (fp) where they cross the midline and turn to travel in the ventral funiculus.
  • Antibody 1C12 also labels the oval bundle (ob) , the dorsal root (dr) and the dorsal root ganglia (drg) , (180X) .
  • FIG. 5 Transmission electron micrograph of the boundary (arrows) between the roof plate (RP) glia and neighboring neurons and neurites in E13.5 rat cervical spinal cord. No neurites cross the roof plate. An example of one of the large extracellular spaces occurs just below the "RP", (7,000X) .
  • FIG. 6 Relationship of the commissural axons to the roof plate (RP) glia in E13.5 rat cervical spinal cord.
  • the commissural axons (ca) , localized with monoclonal antibody 1C12, arise from cell bodies (not stained) along the dorsolateral cord and travel away from the roof plate. Commissural axons do not cross the dorsal midline, (250X) .
  • FIG. 3B An adjacent spinal cord section (low magnification of Fig. 3B) shows the roof plate labelled with an anti-keratan sulfate antibody (a-KS) . Note the absence of reaction product anywhere else in the spinal cord. Superimposition of these two views demonstrates the location of the roof plate glia between, but not overlapping with, the commissural axons; cc, central canal, (250X) .
  • FIG. 7 Differential expression of keratan sulfate epitopes in the roof plate of E15.5 rat cervical spinal cord, localized with various anti-keratan sulfate monoclonal antibodies, (A) a-KS, (B) 4-D-l and (C) 8-C-2 (see Materials and Methods for descriptions) .
  • the roof plate is labelled from the pial surface to the central canal, whereas in (C) , only the dorsal-most portion of the roof plate is immunoreactive.
  • FIG 8. Endo-B-galactosidase and keratanase digestion of the roof plate.
  • A An E13.5 rat cervical spinal cord section treated with chondroitinase ABC for over 2 hours at 37° C, then stained for keratan sulfate with antibody (a-KS) . The labelling pattern is unchanged from that seen when sections are not pre-treated with chondroitinase ABC (compare to Figure 6B) . Skin is also normally stained with this antibody, (400X) .
  • FIG. 9 Other antibodies and a lectin also recognize the roof plate glia but are not unique to this region. These include (A) L2, (B) 5A5 (highly sialylated N-CAM) , (C)a-SSEA-l and (D) lotus lectin (TPA) . Views (A) and (B) are E13.5 and views (C) and (D) are E15.5 rat spinal cord. In (A) , compare the roof plate (RP) , which is L2 immunoreactive ("V-shaped pattern) to the floor plate (fp) , which is entirely devoid of reaction product. Just below this clear region lie the decussating commissural axons.
  • RP roof plate
  • V-shaped pattern V-shaped pattern
  • This antibody also labels the epidermis (e) and the commissural axons (ca) .
  • Antibody 5A5 (B) labels the commissural axons (ca) as well as the roof plate (RP) , among others;
  • a-SSEA-1 (C) labels the roof plate (RP) and the floor plate (not shown) .
  • this antibody like 8-C-2 (Fig. 7C) only recognizes the dorsal-most portion of the roof plate. However, some sections show light labelling with this antibody in the lower portion of the roof plate as well.
  • Lotus lectin (D) labels the roof plate (RP) along the dorsal midline; (A, 160X; B, 180X; C, 400X; D, 400X) .
  • FIG. 10 Localization of cholinesterase in E13.5 and E15.5 rat cervical spinal cord.
  • A The pattern of expression of cholinesterase in E13 spinal cord resembles the pattern of immunostaining for keratan sulfate in the roof plate. Cholinesterase is present in other locations in the cord as well, for example, in the ventricular portion of the basal neuroepithelial cells (be) and the oval bundle of His (ob) (250X) .
  • B on E15.5, the developmentally regulated change in the roof plate morphology coincides with a change in cholinesterase expression. The pattern of expression of cholinesterase is again similar to that of keratan sulfate.
  • FIG. 11 Transverse sections of E15.5 rat spinal cord (compare the roof plate with the anti-KS labelled section in Fig. 12) .
  • the plastic section shows the apical and basal processes of the glial cells and their relationship to the pia and central canal (cc) . Note the proximity of the dorsal column (dc) axons to the roof plate glia, (630X) .
  • FIG. 12 Cryostat sections (10 ⁇ m) of rat cervical spinal cord (E15.5) labelled with an anti-KS antibody (a-KS) .
  • a-KS anti-KS antibody
  • Keratan sulfate epitopes are expressed by other non-innervated regions. Labelling with numerous anti-keratan sulfate antibodies is found (A) and (B) on cells which surround developing rib cartilage in E15.5 rat, and (C) by the outer layer of the epidermis; (A, 250X; B and C, 630X) .
  • FIG. 15 Immunocytochemical labelling of the dorsal midline of optic tectum in hamster mesencephalon with antibodies to keratan sulfate.
  • A Labelling with antibody 4-D-l occurs solely along the tectal midline as shown by horseradish peroxidase reaction product, (400X) .
  • B The tectal midline is also labelled with antibody 8-C-2 shown here in darkfield with immunofluorescence. Note the intensity of reaction product just above the roof plate in the basal lamina with this antibody, (400X) .
  • Other antibodies to keratan sulfate also stain this region (not shown) . Note the dense staining at the ventricle.
  • Substrate preparation technique 60 mm petri dishes are coated with a mixture of methanol and nitrocellulose and air dried in a laminar flow hood. Cellulose strips (350 ⁇ m wide) are soaked in the desired protein solution (e.g. proteoglycan, PG, plus LN or NCAM) + RITC label and transferred to the center of the petri dish in vertical strips (shown by hatched lines) . Laminin is then applied to the entire dish with a bent glass pipet followed immediately by media. Dishes are stored in the dark to preserve fluoroescence until DRGs are dissected and ready for seeding. After 24 hours, the plates are fixed, coverslipped and photographed.
  • desired protein solution e.g. proteoglycan, PG, plus LN or NCAM
  • Strips contain 1 ⁇ g/ml laminin + RITC and 100 ⁇ g/ml laminin is spread over the entire dish.
  • Strips contain 10 ⁇ g/ml laminin + RITC and 100 ⁇ g/ml laminin is spread over the entire dish. Nitrocellulose only binds the first reagent transferred, thus laminin strips result in alternating concentrations of 1 and 100 ⁇ g/ml (A) or 10 and 10 ⁇ g/ml (B) .
  • Arrows denote boundary of lanes in (A) and RITC fluorescence denotes location of lane in (B) . In each case, neurites freely cross the lanes without any signs of inhibition, indicating that neither idiosyncrasies of the protocol nor toxicity of the RITC are problematic in this assay, 250X.
  • Bovine KS/CS-PG (1 mg/ml) + RITC is transferred in strips with 100 ⁇ g/ml laminin spread over the entire dish. Dorsal root ganglia, gently scattered over the center of the dish adhere to the strips of laminin (areas in between strips of KS/CS-PG + RITC) and send out neurites. While abundant growth occurs on laminin, complete inhibition of neurites and support cells occurs when the neurites encounter the KS/CS-PG; 250X.
  • This figure shows 0.2 mg/ml (left) and 0.4 mg/ml (center) .
  • FIG. 20 To determine whether neurites were actively inhibited by 1 mg/ml bovine KS/CS-PG or merely stopping due to the lack of a conducive molecule, we mixed laminin with the proteoglycan.
  • 1.0 mg/ml KS/CS-PG is mixed with 10 ⁇ g/ml laminin + RITC (fluorescence shows location of lanes) .
  • Neurites are still inhibited by KS/CS-PG, even though a concentration of laminin is present which alone allows for abundant outgrowth (see control in Fig. 17) .
  • B When the concentration of laminin is raised to 100 ⁇ g/ml neurites are able to cross the KS/CS-PG containing strip; 250X.
  • Figure 21 Response of DRG neurites to a mixture of l mg/ml KS/CS-PG with polysialylated NCAM at two concentrations: at 10 ⁇ g/ml NCAM, neurites are inhibited by the KS/CS-PG. However, unlike higher concentrations of laminin, 100 ⁇ g/ml NCAM is still inhibitory for all but a few neurites (not shown) ; 4OX.
  • Figure 22 Control for NCAM mixture. Strips containing 10 ⁇ g/ml polysialylated NCAM provide a conducive substrate for neurite outgrowth. Neurites growing from 100 ⁇ g/ml laminin to NCAM in strips show no pattern change or change in fasciculation; 160X.
  • FIG 23 Enzyme digestion assay I.
  • A DRG neurites are inhibited by 1 mg/ml chick KS/CS-PG in the same manner as seen for bovine KS/CS-PG at the same concentration; vertical arrows denote location of lane boundary;
  • B When KS/CS-PG is treated with keratanase, some neurites cross, while many are still inhibited.
  • Vertical arrows denote lane boundary; horizontal arrows point out neurites which have elongated onto the lane; 250X.
  • Figure 24 Enzyme digestion assay II.
  • a rat chondrosarcoma cartilage proteoglycan (RCS; 1 mg/ml) contains chondroitin sulfate, but not keratan sulfate chains.
  • the chondroitin is in the form of C-4-S and not C-6-S like the bovine and chick KS/CS-PG above.
  • This reagent is not a ⁇ effective in the inhibition of neurites as is bovine and chick KS/CS-PG, although partial inhibition can be seen.
  • Vertical arrows denote lane boundary; horizontal arrows point out neurites which have elongated onto the lane; 4OX.
  • Figure 26 In vitro assay for C6 glial cell invasion.
  • A Inhibition of invasion. No cells are found on the strip.
  • B Slight inhibition of invasion. Note the presence of a few cells on the strip, but the cells are not confluent.
  • C No inhibition of invasion. Cell migration and confluence are evident on the strip.
  • the present invention relates to the discovery that keratan sulfate (KS) , chondroitin sulfate (CS) , dermatan sulfate (DS) , heparan sulfate (HS) , heparin (HN) , and/or hyaluronic acid (hyaluronate, HA) can inhibit neurite outgrowth i.e., axonal growth, or nerve regeneration (herein “nerve growth”) or glial cell, in particular astrocyte, migration, invasion or regeneration.
  • KS keratan sulfate
  • CS chondroitin sulfate
  • DS dermatan sulfate
  • HS heparan sulfate
  • HN heparin
  • HA hyaluronate
  • neurite outgrowth i.e., axonal growth, or nerve regeneration (herein “nerve growth") or glial cell, in particular astrocyte, migration
  • Inhibition of neurite outgrowth results from KS and/or CS, DS, HS, HN or HA even in the presence of nerve growth promoting factors such as laminin and NCAM.
  • Inhibition of glial cell, in particular astrocyte, migration or invasion results from KS and/or CS, DS, HS, HN OR HA even in the presence of laminin.
  • the present invention is directed to methods of using KS, and molecules and compositions comprising KS to inhibit or prevent neurite outgrowth and/or glial cell migration or invasion, or nerve or glial cell regeneration, and therapeutically, where the foregoing is desired.
  • Such molecules comprising KS include but are not limited to KS glycosaminoglycan and KS proteoglycan, with keratan sulfate proteoglycan most preferred.
  • the invention is further directed to molecules and compositions comprising CS, and the therapeutic uses thereof to inhibit or prevent neurite outgrowth, glial cell migration or invasion, or nerve or glial cell regeneration.
  • Molecules comprising CS include but are not limited to CS glycosaminoglycan and CS proteoglycan, with chondroitin sulfate proteoglycan preferred.
  • the invention is also directed to molecules and compositions comprising dermatan sulfate and therapeutic uses thereof to inhibit or prevent neurite outgrowth, glial cell migration or invasion, or nerve or glial cell regeneration.
  • Molecules comprising DS include but are not limited to DS glycosaminoglycan and DS proteoglycan, with dermatan sulfate proteoglycan preferred.
  • the invention is further directed to molecules comprising heparan sulfate, heparin, and hyaluronate, and therapeutic uses thereof to inhibit or prevent neurite outgrowth, glial cell migration or invasion, or nerve or glial cell regeneration.
  • inhibitors and antagonists of KS, CS, DS, HS, HN or HA may be used to promote neurite outgrowth or nerve regeneration, i.e., nerve growth, or glial cell, in particular astrocyte, migration, invasion or regeneration and can be administered therapeutically.
  • nerve regeneration i.e., nerve growth, or glial cell, in particular astrocyte, migration, invasion or regeneration and can be administered therapeutically.
  • inhibitors and antagonists include but are not limited to antibodies to KS, CS, DS, HS, HN or HA and derivatives or fragments thereof containing the binding domain, enzymes that degrade KS, CS, DS, HS, HN or HA, lectins specific for KS, CS, DS, HS, HN or HA, and disaccharide antagonists of receptors specific for KS, CS, DS, HS, HN or HA.
  • promotion of neurite outgrowth i.e., axonal growth, or glial cell, in particular astrocyte, migration or invasion, or nerve or glial cell regeneration occurs by removing the inhibitory influence of molecules comprising KS, CS, DS, HS, HN or HA, thus allowing promotion of neurite outgrowth, i.e., axonal growth, or glial cell, in particular astrocyte, migration or invasion, or nerve or glial cell regeneration by endogenous or exogenously added molecules.
  • molecules comprising KS can be used together with molecules comprising another glycosaminoglycan or the disaccharide unit thereof, preferably chondroitin sulfate, in the methods of the invention.
  • the present invention also provides pharmaceutical compositions comprising effective amounts of molecules and compositions comprising KS, CS, DS, HS, HN and/or HA.
  • KS immunocytochemical localization data
  • chondroitin sulfate may be in part responsible for the inhibition of axon elongation through the roof plate in the embryonic spinal cord.
  • keratan sulfate/chondroitin sulfate proteoglycans are actively inhibitory to neurite elongation in a concentration dependent manner.
  • dermatan sulfate inhibits outgrowth of a neuronal cell line and neuronal cells in vitro.
  • a further example in vitro demonstrates that KS/CS-PG and DS-PG inhibit migration and invasion of glial cells and astrocytes.
  • compositions which are envisioned for use in the present invention to inhibit or prevent neurite outgrowth, or glial cell, in particular astrocyte, migration or invasion, or nerve or glial cell regeneration (termed herein “inhibitory compositions") comprise an effective amount of a molecule consisting of at least the disaccharide unit of KS, CS, DS, HS, HN or HA.
  • the molecule can be KS disaccharide, KS glycosaminoglycan, KS proteoglycan, CS disaccharide, CS glycosaminoglycan, CS proteoglycan, DS disaccharide, DS glycosaminoglycan, DS proteoglycan or a compound containing any of the foregoing.
  • such inhibitory compositions include, in addition to such molecules comprising KS, another glycosaminoglycan or proteoglycan or disaccharide unit thereof, selected from the group consisting of such molecules which comprise chondroitin sulfate (CS) and such molecules which comprise dermatan sulfate (DS) .
  • a proteoglycan containing both KS and CS can be used.
  • Both C-4-S and C-6-S sulfur linkage forms of chondroitin sulfate are envisioned as within the scope of the invention, with the C-6-S form being preferred for the inhibition of neurite outgrowth and nerve growth.
  • the KS for use in the present invention includes but is not limited to Type I (corneal) KS (which is unbranched and highly sulfated, and most easily and completely degraded by endo-b-galactosidase and keratanase used sequentially (Melrose and Ghosh, 1985, Anal. Biochem. 170:293-300)) and Type II (skeletal) KS.
  • the molecule may comprise DS.
  • KS/CS-PG may be isolated from the cartilage matrix of cell cultures, such as those of limb mesenchymal cells, by published procedures (see Carrino, A. and Caplan, A. I., 1985, J. Biol. Chem. 260:122-127).
  • KS-PG can be isolated from shark fin, a rich source of KS-PG.
  • KS disaccharide or KS glycosaminoglycan can be isolated after digestion of KS-PG with endo-b-galactosidase or keratanase, respectively (endo-b-galactosidase specifically cleaves between the KS disaccharide residues; keratanase specifically cleaves at the glycosaminoglycan bond to the protein) .
  • KS disaccharides and glycosaminoglycans can be chemically synthesized, or purchased from commercial sources.
  • proteoglycan can be extracted from the cartilage matrix with 4 M guanidinium chloride containing protease inhibitors and purified by CsCl equilibrium density gradient centrifugation and Sepharose CL-2B chromatography (see Haynesworth et al., 1987, J. Biol. Chem. 262:10574- 10581) .
  • the present invention also provides methods of using compositions which promote neurite outgrowth, or glial cell, in particular astrocyte, migration or invasion, or nerve or glial cell regeneration (termed herein "growth-promoting compositions") .
  • growth promoting compositions comprise inhibitors or antagonists or agents which are otherwise destructive (collectively termed herein "growth promoting factors") of the neurite outgrowth and glial cell migration or invasion, or nerve or glial cell regeneration inhibitory activity of keratan sulfate (as exhibited by KS disaccharide, KS glycosaminoglycan, KS proteoglycan, or molecules containing the foregoing) .
  • Such growth promoting factors include but are not limited to antibodies which recognize keratan sulfate, and derivatives and fragments thereof which contain the binding domain, enzymes which degrade keratan sulfate, lectins specific for keratan sulfate, and disaccharide antagonists of the keratan sulfate receptor (see Sections 7.1., 6.3. and 7.2.4.4. , infra) .
  • the growth promoting compositions comprise inhibitors or antagonists or agents which are otherwise destructive of neurite outgrowth, or glial cell migration or invasion, or nerve or glial cell regeneration inhibitory activity of CS (as exhibited by CS disaccharide, CS glycosaminoglycan, CS proteoglycan, or molecules containing the foregoing) .
  • the invention is also directed to antibodies to CS (and fragments thereof), enzymes which degrade CS, lectins specific for CS, and disaccharide antagonists of the CS receptor.
  • the growth promoting compositions comprise inhibitors or antagonists or agents which are otherwise destructive of neurite outgrowth, or glial cell migration or invasion, or nerve or glial cell regeneration inhibiting activity of DS (as exhibited by DS disaccharide, DS glycosaminoglycan, DS proteoglycan, or molecules containing the foregoing) .
  • DS disaccharide
  • DS proteoglycan as exhibited by DS disaccharide, DS glycosaminoglycan, DS proteoglycan, or molecules containing the foregoing
  • the invention is also directed to antibodies to DS (and fragments thereof) , enzymes which degrade DS, lectins specific for DS, and disaccharide antagonists of the DS receptor.
  • the growth promoting compositions comprise inhibitors or antagonists or agents which are otherwise destructive of neurite outgrowth, or glial cell migration or invasion, or nerve or glial cell regeneration inhibiting activity of HS, HN or HA (as exhibited by HS, HN or HA disaccharide, HS, HN or HA glycosaminoglycan, or HS, HN or HA proteoglycan, or molecules containing the foregoing) .
  • the invention is also directed to antibodies to HS, HN or HA (and fragments thereof) , enzymes which degrade HS, HN or HA, lectins specific for HS, HN or HA, and disaccharide antagonists of the HS, HN or HA receptor.
  • Antibodies which recognize keratan sulfate, CS, DS, HS, HN or HA, and which may be used, include previously isolated known antibodies as well as antibodies which can be newly generated.
  • KS disaccharide, KS glycosaminoglycan, KS-PG, or compositions comprising the same may be used as an immunogen to generate anti-KS antibodies.
  • CS disaccharide, CS glycosaminoglycan, CS-PG, or compositions comprising same may be used as an immunogen to generate anti-CS antibodies.
  • DS disaccharide, DS glycosaminoglycan, DS-PG, or compositions comprising the same may be used as an immunogen to generate anti-DS antibodies.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to epitopes of KS, CS, DS, HS, HN or HA.
  • KS, CS, DS, HS, HN or HA-containing compositions 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 polyol ⁇ , polyanions, peptides, oil emul ⁇ ion ⁇ , keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-
  • a monoclonal antibody to KS, CS, DS, HS, HN or HA is produced.
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other specie ⁇ ) monoclonal antibodie ⁇ .
  • 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. Enzy ol. 92:3-16).
  • Chimeric 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).
  • Previously prepared monoclonal antibodies to KS which may be used according to the present invention include but are not limited to antibodies MZ15 (Zanetti et al., 1985, J. Cell Biol. 101:53-59) (specific for sulfated poly N-acetyllactosamine domain ⁇ on KS) ; 1/20/5-D-4 (Caterson et al., 1983, J. Biol. Chem. 258:8848-8854); 4/8/1-B-4 (Caterson et al., 1985, Fed. Proc.
  • Monoclonal antibody 3-B-3 specifically recognizes the C-4-S form of chondroitin ⁇ ulfate after dige ⁇ tion of the C-6-5 form by chondroitin ABC lyase (Couchman, J. R. , 1984, Nature 307:650-652) .
  • a molecular clone of an antibody to a KS, CS, DS, HS, HN or HA 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, New York) may be u ⁇ ed to con ⁇ truct nucleic acid sequences which encode a monoclonal antibody molecule, or antigen binding region thereof.
  • Antibody molecules may be purified by known techniques, e.g., immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography) , or a combination thereof, etc.
  • Antibody fragments which contain the binding domain of the molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab , )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')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • Enzymes that degrade keratan sulfate can be used in the practice of the instant invention, and include but are not limited to endo-b-galactosidase and keratanase. In a specific embodiment, both endo-b-galactosidase and keratanase can be used, simultaneously or sequentially, to degrade KS.
  • enzyme( ⁇ ) that degrade KS can be u ⁇ ed ⁇ i ultaneously or sequentially with enzyme( ⁇ ) that degrade another proteoglycan/glycosaminoglycan, e.g. enzymes that degrade chondroitin sulfate or dermatan sulfate.
  • the enzyme that degrades chondroitin sulfate is chondroitin ABC lyase.
  • Endo-b-galactosidase, keratanase, and chondroitin ABC lyase are commercially available (e.g., Miles Scientific) .
  • enzymes that degrade CS can be used in the practice of the invention, and include but are not limited to chondroitinase and chondroitin ABC lyase.
  • enzyme ⁇ that degrade DS can be used in the practice of the invention, and include but are not limted to chondroitin ABC lyase.
  • enzymes that degrade heparan sulfate, heparin, or hyaluronate can be used. These enzymes include, but are not limited to, heparanase and hyaluronidase. 5.2.3. OTHER COMPOSITIONS
  • Lectins al ⁇ o referred to as agglutinins, ⁇ pecific for KS, CS, DS, HS, HN or HA compri ⁇ e another aspect of the growth promoting composition ⁇ of the invention.
  • Lectin ⁇ that bind to keratan sulfate can be used in the practice of the instant invention.
  • lectins that bind chondroitin sulfate can be used in the practice of the invention.
  • lectins that bind dermatan sulfate can be used in the practice of the invention.
  • lectins specific for heparan ⁇ ulfate, heparin or hyaluronate can be u ⁇ ed in the practice of the invention.
  • the lectin from triticum vulgaris (wheat germ) specific for N-acetyl-D- glirco ⁇ amine can be u ⁇ ed.
  • the triticum vulgari ⁇ lectin bind ⁇ to KS, CS and DS.
  • the tetrogonolobu ⁇ purpurea ⁇ agglutinin (TPA) may be used.
  • Other lectins useful in the practice of this invention include, but are by no means limited to, lectins from abrus precatoriu ⁇ (Jequirity bean agglutinin) , arachi ⁇ hypogea (peanut agglutinin) , bandeiraea ⁇ implicifolia, erythrina corallodendron (Coral tree agglutinin) , helix pomatia (Roman snail agglutinin) and helix aspersia (garden snail aglutinin) , limulus polyphemu ⁇ (limulin or hor ⁇ e ⁇ hoe crab agglutinin) , maclura pomifera, (osage orange agglutinin) momordica charantia, phaseolus limensis (lima
  • Disaccharide antagonists that block receptors on nerve or glial cells specific for KS, CS, DS, HS, HN or HA can also be used in the practice of the instant invention. Suitable disaccharide antagonists bind to the receptor for, but do not effect the inhibitory functions of KS, CS, DS, HS, HN or HA.
  • Metabolic blockers of proteoglycan synthesi ⁇ may also be used in the practice of the invention. Drugs or agents that inhibit or prevent synthesis or secretion of proteoglycan ⁇ or glyco ⁇ aminoglycans prevent the synthesis or secretion of KS, CS, DS, HS, HN or HA, and thus preclude the inhibitory effects of KS, CS, DS, HS, HN or HA.
  • the inhibitory compositions of the invention can be therapeutically useful where an inhibition of neurite outgrowth, glial cell migration or invasion, or nerve or glial cell regeneration is desirable.
  • an inhibitory composition can be used in the treatment of patients with gliomas or tumor ⁇ of nerve ti ⁇ sue, e.g., malignant tumors such as a neuroblastoma.
  • an inhibitory composition can be u ⁇ ed for the treatment of a neuroma (undirected axon growth a ⁇ ociated with situations where the axon is missing either its appropriate target or substrate pathway for neural development) .
  • treatment of neuroma associated with amputation, lesion, or congenital deformities, etc. can be treated.
  • Disorders resulting from an overproduction of nerve growth-promoting factors can also be treated by administration of an inhibitory compo ⁇ ition.
  • the inhibitory compo ⁇ ition ⁇ can be u ⁇ ed to treat di ⁇ orders of the central and/or peripheral nervous sy ⁇ tem ⁇ .
  • the products of this invention can be used as barriers to glial cell migration or invasion caused by trauma, surgery, infection (viral or bacterial) , metabolic disease, malignancy, exposure to toxic agents, or other hyperplastic situations. They may be used specifically to protect an organ or tissue from the previously mentioned conditions through a coating procedure.
  • dorsal root ganglia, optic nerve, and optic chiasma may be coated with proteoglycan ⁇ in order to protect again ⁇ t uncontrolled cell inva ⁇ ion and adhe ⁇ ion.
  • Thi ⁇ may be useful as a preventitive or prophylactic treatment or may be applied as a treatment in patients where a disorder has already been manifested.
  • composition ⁇ including keratan sulfate, in any molecular form in which it may be made or found, either alone or with chondroitin sulfate, which also may be in any molecular form in which it may be found, or dermatan sulfate, which al ⁇ o may be in any molecular form in which it may be found can be u ⁇ ed to preferentially inhibit neurite outgrowth.
  • keratan ⁇ ulfate and/or chondroitin ⁇ ulfate or dermatan ⁇ ulfate, in any molecular form in which it may be found may be u ⁇ ed to preferentially inhibit glial cell, in particular astrocyte, migration or invasion.
  • the growth-promoting compositions of the invention can be used therapeutically in regimens where neurite outgrowth is inhibited and an increase in neurite outgrowth or nerve regeneration is desired, e.g., in patients with nerve damage, or in regimens where glial cell, in particular astrocyte, migration, invasion or regeneration is desired.
  • the growth-promoting compositions can be administered to patients in whom nerves or glial cells have been damaged by trauma, surgery, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, toxic agents, paraneoplastic syndromes, stroke, degenerative disorders of the nervous system, etc.
  • ⁇ uch di ⁇ order ⁇ examples include but are not limited to Alzheimer-'s Disease, Parkinson-'s Disease, Huntington's chorea, amyotrophic lateral sclerosis, progressive supranuclear palsy, and peripheral neuropathies.
  • the growth-promoting compositions can be therapeutically applied ⁇ o a ⁇ to allow access to the sites of amyloid plaques (see Selkoe, D. J., 1989, Cell 58:611- 612) .
  • the growth- 5 promoting compositions of the invention can be used to promote nerve growth through an exi ⁇ ting scar or a scar in the proces ⁇ of formation.
  • the growth-promoting compositions may be used in the central and/or peripheral nervous sy ⁇ tems, e.g., to prevent the inhibition of and 0 thus promote the regeneration of nerve pathways, fiber systems and tracts.
  • growth promoting and/or inhibitory composition ⁇ of the invention may be used _ to appropriately direct axon growth along desired paths.
  • the growth promoting compositions of the invention may be used to promote the migration or invasion of glial cells, in particular astrocyte ⁇ .
  • compositions which comprise an effective amount of an an inhibitory composition or a growth- 5 promoting composition, as the case may be, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including, but not limited to, saline, buffered saline, dextro ⁇ e, and water.
  • the amount of inhibitory or growth-promoting compo ⁇ ition which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • a high concentration of the molecule co pri ⁇ ing KS, CS, DS, HS, HN or HA relative to the concentration of factors which promote neurite outgrowth or adhesion (e.g. laminin, NCAM)or glial cell, including astrocyte, migration or invasion at the desired site of therapy is preferred for use.
  • Methods of introduction of the pharmaceutical compo ⁇ itions of the invention include methods known to those skilled in the art. It may be desirable to introduce the pharmaceutical compo ⁇ itions of the invention into the central nervou ⁇ ⁇ y ⁇ tem by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, ⁇ uch a ⁇ an Ommaya re ⁇ ervoir.
  • compositions of the invention may be administered locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during ⁇ urgery, by injection, by mean ⁇ of a catheter, or by mean ⁇ of an implant, said implant being of a porou ⁇ , non- porous, or gelatinous material, including membrane ⁇ , ⁇ uch a ⁇ sialastic membranes, or fibers.
  • Polymer implants coated with the pharmaceutical composition can be applied or inserted at the desired site of treatment. Such polymers can have various compositions, pore sizes, and geometries.
  • Polymers which can be used include but are not limited to tho ⁇ e made of nitrocellulo ⁇ e, polyanhydride ⁇ , and acrylic polymers.
  • the invention also provides for the pharmaceutical compositions to be administered via liposomes, icroparticles, microcapsules, or other semipermeable membranes.
  • a recombinant cell secreting an enzyme that degrades KS, CS, or DS can be administered where a growth-promoting compo ⁇ ition i ⁇ indicated.
  • a hybridoma cell ⁇ ecreting an anti-KS, anti-CS or anti-DS monoclonal antibody can be admini ⁇ tered where a growth- promoting compo ⁇ ition is indicated.
  • the cells may be encapsulated in a suitable biological membrane and implanted in the patient.
  • MOLECULAR AND CELLULAR CHARACTERIZATION OF THE GLIAL ROOF PLATE OF THE SPINAL CORD AND OPTIC TECTUM A ROLE FOR KERATAN SULFATE PROTEOGLYCAN IN THE DEVELOPMENT OF AN AXON BARRIER
  • Certain types of glial structure ⁇ located at ⁇ trategic positions along the edges of axon pathways, may provide the mechanical and/or chemical elements for the construction of barriers which can grossly direct the elongation of axons during development.
  • the roof plate a putative axon barrier, is located along the dorsal midline of the developing spinal cord and may be important for the guidance of the commissural and dorsal column axons.
  • a molecule which i ⁇ unique to the roof plate when axon ⁇ grow close to, but do not cross, the dor ⁇ al midline is a glycosaminoglycan (GAG) , keratan sulfate. Keratan sulfate is also present in the tectal midline and in other non-innervated region ⁇ ⁇ uch a ⁇ the outer epidermis and developing cartilage.
  • GAG glycosaminoglycan
  • the trunk region of Sprague-Dawley rat embryos, day 11.5 (Ell.5), E12.5, E13.5, E14.5 and E15.5 were fixed by immersion in 4% paraformaldehyde/1% glutaraldehyde in 0.15 M phosphate buffered saline (PBS) overnight at 4°C.
  • PBS phosphate buffered saline
  • the sections were washed for an additional hour in 0.15M PBS, dehydrated in ethanol and embedded in Spurr's resin.
  • One micron sections were stained with 1% toluidine blue. Thin sections were stained with uranyl acetate and lead citrate for electron microscopy.
  • the salt concentration of the buffer was varied by 1.5-2.0 to observe the effect on the extracellular spaces between the roof plate gli
  • Monoclonal antibody 1C12 (Dodd et al., 1988, Neuron, 1:105-116) recognizes a glycoprotein on commissural axons.
  • Antibody 5A5 bind ⁇ to the poly ⁇ ialic acid moietie ⁇ on NCAM. It has been shown by other ⁇ that NCAM may be the only source of poly ⁇ ialic acid in chick brain. Endo-N-treatment of NCAM to remove ⁇ ialic acid re ⁇ ult ⁇ in a lack of NCAM recognition by antibody 5A5. Further, 5A5 labels a band on a Western blot of brain at 250 kD which correspond ⁇ to the molecular weight of highly sialylated NCAM.
  • Anti-SSEA-1 (Solter, D. , and Knowles, B. B., 1978, Proc. Natl. Acad. Sci. U.S.A. 75(11) :5565-5569) recognizes a stage-specific embryonic antigen which is fir ⁇ t expressed in blasto eres of 8-cell stage mouse embryos.
  • the monoclonal antibody MZ15 (Zanetti et al.,
  • Monoclonal antibody a-KS is specific to an epitope of keratan sulfate.
  • L2 Koreane et al., 1984, Nature (London) 311:153-155
  • Melitta Schachner Sewiss Federal Institute of Technology, Zurich
  • Enzyme treatment consisted of incubating the tis ⁇ ue ⁇ ection ⁇ for 20, 40 or 60 minutes with a concentration of 0.001, 0.01 or 0.1 unit/ml of either endo-B-galactosidase or keratanase or both in sequence.
  • the block was removed and the sections were incubated in an HRP conjugate of the winged or asparagu ⁇ pea lectin, tetragonolobus purpureas agglutinin (TPA) , also known as lotus tetragonolobus or lotus lectin (Steindler, D. A., and Cooper, N. G. F., 1987, Dev. Brain Res. 36:27- 38) overnight at 4° C. at a concentration of 1:75 or 1:100. TBS/BSA with cations was added to the primary incubation to facilitate TPA binding. The tis ⁇ ue was washed and the TPA visualized with diaminobenzidine (see protocol in Section 6.1.2). Following a final wash, the tis ⁇ ue wa ⁇ dehydrated and cover ⁇ lipped a ⁇ above.
  • TPA tetragonolobus purpureas agglutinin
  • CHOLINESTERASE ASSAY Embryonic day 13.5 and E15.5 rat ⁇ were immer ⁇ ion fixed in 4% paraformaldehyde in 0.1 M PBS and the ⁇ pinal cord ⁇ were cryo ⁇ tat ⁇ ectioned (10-15 lm) .
  • the ti ⁇ ue sections were processed for cholinesterases using a modification of Koelle, G. B. , and Friedenwald, J. S., 1949, Proc. Soc. Exp. Biol. Med. 70:617-622.
  • the sections were rinsed in distilled water numerous times and incubated overnight at room temperature in the dark in a mixture of 0.05 M sodium acetate, 4 mM copper sulfate, 16 mM glycine and acetylthiocholine iodide.
  • the sections were rinsed and incubated in 1% sodium sulfide for 5-10 minutes, rinsed again and incubated in 4% formalin buffer overnight at 4° C.
  • the ti ⁇ sue was rinsed a final time, dehydrated and coverslipped a ⁇ above.
  • the roof plate undergoes morphological changes between embryonic day 11.5 (Ell.5) and E12.5.
  • the cells of the roof plate are arranged in an arching pattern in comparison to adjacent neuroepithelial cells which are more radial (Fig. 2A) .
  • the extracellular space between the roof plate cells is minimal and comparable to that between the adjacent cells.
  • large extracellular spaces about 2-10 ⁇ m in diameter, can be seen between the primitive roof plate glia but not between the adjacent cells (Fig. 3A) .
  • the large ⁇ ize and ⁇ hape of the ⁇ paces are consistent in all animals and they are located preferentially along the apical region of the roof plate.
  • the roof plate cells are arranged in the shape of a "wedge". With the electron microscope, we have ob ⁇ erved that the apical processes of the roof plate cells terminate at the pial surface in endfeet and the basal proce ⁇ e ⁇ appear to end at the dorsal central canal. Not every cell in the roof plate spans from the pial surface to the central canal, as these cells are dividing until E14 (Altman, J., and Bayer, S. A., 1984, in Advances in Anatomy, Embryology and Cell Biology, Vol. 85, Springer-Verlag, Heidelberg, Germany, pp. 53-83) .
  • the roof plate is approximately 70 ⁇ m long from the pia to the central canal at E13.5 and about 100 ⁇ m wide at the midpoint.
  • Rostral-caudal analyses of 1 ⁇ m plastic sections indicate that the extracellular space ⁇ are present in the roof plate throughout the cervical and thoracic spinal cord.
  • the spaces appear to be actual and not due to processing of the tis ⁇ ue ⁇ ince variou ⁇ perturbations, e.g. varying the salt concentrations of the buffers by a factor of 1.5-2.0 does not alter the spaces relatively more or les ⁇ then those in the surrounding tissue.
  • Altman and Bayer (1984, supra) have also observed large caliber extracellular spaces in the roof plate in tissue prepared differently.
  • Two axon sy ⁇ tem ⁇ are present in the dorsal region of the spinal cord and travel near the roof plate at times which seem appropriate for the roof plate to act as a barrier to them.
  • the proces ⁇ e ⁇ of these neurons can be vi ⁇ ualized with antibody 1C12 (Dodd et al., 1988, Neuron 1:105-116) (Fig. 4).
  • these axon ⁇ re ⁇ ide in the oval bundle about 150 ⁇ from the dor ⁇ al midline (Fig. 1) .
  • E15 they abut the roof plate at the dorsal midline of the spinal cord.
  • Ultrastructural observation ⁇ show that neurites do not cross the roof plate but are found in close apposition to it all along its perimeter (Fig. 5) . Thu ⁇ , it appears that all processes from cells adjacent to the roof plate are excluded from this dorsal midline structure.
  • FIG. 6a and 6B depict the relationship of the keratan sulfate labelling of the roof plate cells to nearby commis ⁇ ural axons labelled with 1C12.
  • keratan ⁇ ulfate expression appears well before the arrival of the dorsal column axons and seem ⁇ not to be pre ⁇ ent prior to, but rather, at about the ⁇ ame time that the dorsal-most commis ⁇ ural population is extending axons.
  • the ⁇ e markers demonstrate that keratan sulfate epitopes are specific to the roof plate and are found nowhere else in the spinal cord at this stage of development (Fig. 6B) .
  • Antibodies a-KS and 4-D-l label the dorsal midline from the dorsal-most to the ventral-most portion (Figs. 7A and B) , while antibodie ⁇ 8-
  • anti-keratan sulfate antibodies also label other structures in the sections we studied.
  • many of the anti-keratan sulfate antibodies label epidermis (Figs. 7B and C) , which has been shown by Funderburgh et al. (1986, Dev. Biol. 116:267-277) to contain this glycosaminoglycan.
  • Antibody 8-C-2 additionally labels the ba ⁇ al lamina ⁇ urrounding the spinal cord (Fig. 7C) .
  • keratan sulfate-containing tis ⁇ ues such as cartilage and basal lamina retained much of their keratan sulfate expres ⁇ ion following enzymatic digestion, although skin showed some observable decrease in staining intensity.
  • the controls showed that chondroitinase digestion had no visible effect on the intensity of keratan sulfate immunostaining of the roof plate (Fig.
  • the primitive roof plate glia express a number of other characteristic molecules on E13.5, but in contrast to keratan sulfate, these are seen elsewhere in the spinal cord.
  • the carbohydrate recognized by monoclonal antibody L2/HNK-1 (glucuronic acid 3-sulfate) is expressed by the roof plate cells (Fig. 9A) .
  • the floor plate is entirely devoid of labelling with L2.
  • Antibody 5A5 localizes highly sialylated NCAM to only the midline portion of the roof plate (Fig. 9B) . Both L2 and 5A5 label the DRG, the dorsal and ventral roots, the dorsal root entry zone and the entire marginal zone of the spinal cord.
  • FIG. 10A A histochemical a ⁇ ay for choline ⁇ terase (ChE) showed that the roof plate glia express this molecule as well on E12.5 and E13.5 (Fig. 10A) .
  • the pattern of ChE expression on E12.5 and E13.5 in the roof plate is like that of the anti-keratan sulfate antibodies at this age, i.e. in a wedge-shaped distribution.
  • ChE staining is also present in the dorsal root entry zone, on glial cells of the sulcus limitan ⁇ and the ventricular portion of the ba ⁇ al plate neuroepithelia at this time (Fig. 10A) .
  • the roof plate undergoe ⁇ a second and more dramatic morphological alteration by E15.5.
  • the presumptive glial cells become transformed into a long, thin septum-like structure in the dorsal midline (Fig. 11) .
  • the extracellular spaces are greatly diminished, resulting in a denser construct than that seen before this age.
  • the roof plate spans approximately 160 ⁇ from the pial surface to the top of the central canal, but is only 10-15 ⁇ m wide, except at its dorsal aspect where it widens. Thus, the roof plate has undergone about a two-fold increase in length and approximately a ten-fold decrease in width.
  • Keratan ⁇ ulfate epitope ⁇ were expressed by developing cartilage. Label is pre ⁇ ent surrounding groups of chondrocytes (Fig. 13) and in ⁇ ome ca ⁇ e ⁇ around the individual chondrocyte ⁇ them ⁇ elves.
  • TPA labels the doral midline in its entirety from the pial ⁇ urface to the dorsal central canal (Fig. 9D) .
  • ChE is present along the dorsal midline from the pial surface to the top of the central canal on E15.5 (Fig. 10B and C) .
  • it is also expres ⁇ ed by a subpopulation of sen ⁇ ory axon ⁇ , in the ventricular portion of the ba ⁇ al neuroepithelia, the motor cells, the ventral root and in the sulcus limitans, as well as in the developing limb bud cartilage of the upper trunk.
  • the roof plate occupies a minimal area of the dorsal midline laterally but still spans from the pial surface to the dorsal central canal. Keratan sulfate epitopes are no longer detectable with immunocytochemistry in the roof plate (Fig. 14) nor in the basal lamina surrounding the spinal cord at E17.5, but they persist in cartilage and epidermis.
  • the roof plate of the spinal cord undergoes morphological and molecular changes during early embryonic development.
  • a network of large extracellular space ⁇ develop ⁇ near the pial ⁇ urface between the glial cell ⁇ of the roof plate and contribute ⁇ to the roof plate's wedge shape.
  • the shape of the roof plate has changed to a long, thin septum at the midline and the amount of extracellular space is significantly reduced.
  • the roof plate cells expres ⁇ a number of molecules which are also present in other regions of the spinal cord, a particular glycosaminoglycan, keratan sulfate, i ⁇ expre ⁇ sed solely by the roof plate glia beginning on E12.5 and is no longer detectable by E17.5.
  • roof plate interact ⁇ specifically with, exerting its inhibitory influence during development on, axons that elongate near the midline, i.e. a small subpopulation of the ventral commissural system and a larger number of axons which constitute the medial-mo ⁇ t (i.e. gracile tract) fibers of the dorsal columns.
  • keratan sulfate doe ⁇ not occur alone in vivo but rather a ⁇ a keratan sulfate/chondroitin sulfate proteoglycan (KS/CS-PG) .
  • KS/CS-PG keratan sulfate/chondroitin sulfate proteoglycan
  • chondroitin sulfate as well as other glycosaminoglycans and/or proteoglycans may be present in the roof plate and may be acting in combination with other molecules such as keratan sulfate glycosaminoglycan/proteoglycan to generate axon inhibition.
  • glial structures may act as axon barriers or boundaries in regions of the nervous sy ⁇ tem other than the roof plate.
  • the "knot-like 1 * structure which is suggested (Silver, J.
  • the migrating fibers choose one of the functionally advantageous pathways toward the midbrain and diencephalon instead of turning rostrally to enter the olfactory region of the telencephalon.
  • roof plate of the developing spinal cord functions similarly to the midline glial structure of the tectum, it may constitute an e ⁇ ential blockade to aberrant axon elongation.
  • barriers at the dorsal midline of the central nervous system may be instrumental in separating right versus left side sensory information.
  • Glia may also ⁇ erve to compartmentalize regions of axonal arborization.
  • a type of boundary glia identified by anti-glial fibrillary acidic protein (GFAP) has been observed in neonatal cortex (Cooper, N. G. F. , and
  • Glial cells of the barrel wall domains apparently reflect the mature patterning of the thalamic terminal arbors related to vibrissal function. However, this form of boundary differs from the roof plate. It appears to be more plastic since the intense matrix producing-glia of the barrel walls are able to shift their position geometrically in response to an activity-dependent signal associated with the afferent axons. Cells that may play a similar role in cordoning synaptic territories have also been observed by Oland et al. (1988, J. Neurosci. 8(1) :353-367) in the olfactory region of the moth, Manduca sexta.
  • the roof plate cells of the spinal cord deserve discussion on three separate but interrelated aspects which may provide evidence about their shape and mechanism of axon repulsion: (1) the possible structural contribution of the extracellular space between the glial cells, (2) the absence of growth of axons through the extracellular spaces and (3) the inhibitory functions of the molecules expressed by these cells.
  • Keratan sulfate epitopes may function, in part, in the creation of a molecular barrier in the roof plate. Also, we have observed with light microscopy that the region surrounding developing cartilage and the matrix around individual chondrocytes expre ⁇ keratan ⁇ ulfate-like immunoreactivity. Cartilage i ⁇ not innervated. Further, we have ⁇ hown that outer epidermis expresses keratan sulfate epitopes during development.
  • tissue culture experiments demonstrate that keratan sulfate glycosaminoglycans can directly inhibit axon growth (see Section 7 infra) .
  • Enzymatic digestion of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycosaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition.
  • Cellulose (Whatman Filter paper, #1) was cut into 350 ⁇ m strips and used to blot various protein substances down onto the nitrocellulose substrate. Each protein solution contained either rhodamine isothiocyanate (RITC) or fluorescein isothiocyanate (FITC) as a marker which could later be detected to determine the exact position of the ⁇ trip ⁇ .
  • RITC rhodamine isothiocyanate
  • FITC fluorescein isothiocyanate
  • the cellulo ⁇ e ⁇ trips were soaked in 20 ⁇ l of the desired protein solution, transferred to the nitrocellulose-coated dish in a vertical pattern (FIG. 1) , allowed to set for 30 second ⁇ then removed.
  • Chick E9 dorsal root ganglia were dissected in a calcium-magnesium free buffer by decapitating the chick, eviscerating, then carefully removing the vertebral column and spinal cord. The DRGs were then cleaned free of surrounding tissue and plucked out using fine forceps. The media in the test culture dishes was removed and replaced with fresh media containing
  • the DRGs were picked up and scattered gently around the center of the dish containing the patterned stripes. Approximately 20 DRGs were seeded onto each dish. The dishes were then incubated for 24 hours followed by fixation with 4% paraformaldehyde/0.1% glutaraldehyde for 1 hour. The dishes were coverslipped in
  • Each well was filled with a different anti-keratan sulfate antibody: 8-C-2 and 4-D-l, a-KS, 5-D-4 and l-B-4 (Caterson et al., 1985, Fed. Proc. 44:386-393) or MZ15 (Zanetti, et al., 1985, J. Cell Biol. 101:53-59), 1:100 in a mixture of 10 mM PBS + 3% NGS + 0.2% Triton X-100 and incubated at 37°C overnight.
  • the wells were rin ⁇ ed 5X with buffer, and a goat anti-mouse HRP-conjugated IgG or IgM secondary antibody wa ⁇ added and incubated overnight at 37°C.
  • the nitrocellulose paper was then reacted with 0.01% . diammobenzidme in PBS + 0.003% hydrogen peroxide. All dots showed a reaction product indicating that the KS/CS-PG was bound to the paper.
  • proteoglycan ⁇ were extracted with 4 M guanidiniu chloride containing protea ⁇ e inhibitor ⁇ and purified by C ⁇ Cl equilibrium den ⁇ ity gradient centrifugation and Sepharose CL-2B chromatography
  • the appropriate fractions from the Sepharose CL-2B column were pooled, dialyzed against distilled water at 4°C and lyophilized to drynes ⁇ ⁇ o that the number of 35S cpm/mg dry weight could be determined.
  • the area of the culture dish containing the bound proteoglycans was excised and transferred to a 20-ml glass scintillation vial.
  • Cytoscint ⁇ cintillation cocktail (ICN) wa ⁇ added to the vial and the amount of bound 35S wa ⁇ determined by ⁇ cintillation ⁇ pectrometry on a Beckman LS 6800 counter. From the amount of bound 35S, the amount of bound proteoglycan could be calculated based on the number of 35S cpm/mg dry weight.
  • KS/CS-PG (1 mg/ml) + LN (10 ⁇ g/ml) .
  • LN 10 ⁇ g/ml
  • concentration of the KS/CS-PG was maintained at 1 mg/ml, which we knew to produce maximum inhibition of neurites. The remainder of the experiment proceeded as above. Controls consisted of strips of 10 ⁇ g/ml laminin + RITC and
  • KS/CS-PG:NCAM MIXTURES Polysialylated NCAM is present in the roof plate during development (see Section 6, supra) .
  • PG and NCAM gifts of P. Yang and U. Rutishau ⁇ er
  • Polysialylated NCAM was prepared by immunoaffinity purification in milligram quantities from the 0.5% NP40 extracts of E14 chick brain vesicles.
  • NCAM in the extracts binds to anti-chick NCAM monoclonal antibody (5E) IgG conjugated to Sepharose 4B beads which are activated by the cyanogen bromide method, and NCAM is eluted with 0.57% diethylamine, pH 11.5 (Hoffman et al., 1982, J. Biol. Chem. 257(13) : 1120-1129) .
  • the resulting NCAM is poly ⁇ ialylated and runs above 200 kD on SDS polyacrylamide gels.
  • NCAM (10 or 100 ⁇ g/ml) wa ⁇ u ⁇ ed in combination with 1 mg/ml KS/CS-PG and the incubation ⁇ conducted a ⁇ above. The ⁇ e di ⁇ hes were compared to the KS/CS-PG + LN mixtures and to the KS/CS-PG alone.
  • thi ⁇ concentration was observed to significantly degrade the keratan sulfate in 10 lm frozen sections of rat spinal cord.
  • the DRGs were seeded and the cells were incubated for 24 hours as done previously. Dishes containing the enzyme treated stripes were incubated simultaneously with control dishes which did not receive enzyme and neurite outgrowth was compared.
  • Certain control dishe ⁇ included protea ⁇ e inhibitor ⁇ (1 mg/ml each of apoprotin, leupeptin and pepstatin in 10 mM
  • Tris-acetate buffer pH 7.2
  • This proteoglycan is much like the bovine or chick KS/CS-PG except that it lacks the KS chain region and the chondroitin sulfate is in the C-4-S form rather than the C-6-S form found in bovine and chick KS/CS-PG.
  • DS- PG dermatan sulfate proteoglycan
  • nitrocellulose-coated culture di ⁇ he ⁇ as a substrate by which to attach such proteins a ⁇ laminin (LN) and neural cell adhe ⁇ ion molecule (NCAM) , which are known to facilitate cell attachment and/or allow for the elongation of neurite ⁇ (Ruti ⁇ hauser et al., 1978, J. Cell Biol. 79:382-393; Letourneau, P. C, 1975, Dev. Biol. 44:92-101; Manthorpe et al., 1983, J. Cell Biol.
  • LN laminin
  • NCAM neural cell adhe ⁇ ion molecule
  • KS/CS-PG variou ⁇ portions of this macromolecule (it ⁇ protein core, or KS-PG, or CS-PG) .
  • DS-PG dermatan sulfate proteoglycan
  • RCS cartilage proteoglycan
  • KS/CS-PG LN MIXTURES
  • the KS/CS-PG was bound to the nitrocellulose in the dishe ⁇ , ba ⁇ ed on po ⁇ itive re ⁇ ult ⁇ of a dot blot immunoa ⁇ ay for keratan sulfate, however, it was unknown whether all of the stripe region was covered with the proteoglycan, or whether there was space left for the laminin to bind (either to the nitrocellulose coating or perhaps even to the proteoglycan itself) when the dish was covered sequentially first with the PG then with LN.
  • KS/CS-PG:NCAM MIXTURES In the above assay, LN was used as a stimulatory molecule for adhesion and elongation. However, based on immunos ining by us and others, we believe that, although LN in its extracellular form is present adjacent to the roof plate in the lateral walls of the spinal cord, it may be present only in very low concentrations or only in the cytoplasmic form within the roof plate itself. Therefore, we tested a molecular combination in the stripe assay using KS/CS-PG + polysialylated NCAM, shown previously to be expres ⁇ ed by the roof plate cells (Section 6, supra) .
  • this CS-PG molecule is less effective than the bovine or chick KS/CS-PG in achieving DRG neurite inhibition. Since partial inhibition was observed, the data indicated that not only may the KS chains play a role 5 in complete inhibition, but that CS can also be a major contributor to the repulsion of neurites. Consideration must be given here to the fact that neurite outgrowth in response to C-4-S in the RCS and C-6-S of the bovine and chick KS/CS-PG cannot be directly compared. 0
  • the roof plate also expresse ⁇ KS which we have shown in this in vitro ⁇ tudy to be inhibitory to DRG neurite ⁇ .
  • KS which we have shown in this in vitro ⁇ tudy to be inhibitory to DRG neurite ⁇ .
  • the glial cells of the roof plate in vivo are capable of simultaneously producing or sequestering molecules for cell-cell attachment as well as for cell repulsion.
  • This scenario may also occur at other axon refractory sites in the CNS such a ⁇ the chick ⁇ ub-plate where large extracellular spaces bordered by glial cell processes and filled with CS proteoglycan-containing extracellular matrices have been de ⁇ cribed (Palmert et al., 1986, Society for Neuro ⁇ ci. Ab ⁇ t. 12:1334).
  • the RCS proteoglycan consists of chondroitin sulfate in the form of
  • glycosaminoglycans produce varying amounts of inhibition, dependent upon concentration and type
  • glycosaminoglycans can be made to be growth permissive, i.e., the inhibitory effect of the proteoglycan can be reduced or completely masked if accompanied by an appropriate concentration of the growth-promoting molecule, laminin.
  • NCAM was far less effective in counteracting the proteoglycan-mediated inhibition. The ⁇ e re ⁇ ult ⁇ ⁇ uggest that a growth cone can sample chemical differences in it ⁇ environment and make motile "deci ⁇ ions" based on summation of its sampling (see Letourneau, P. C. , 1975, Dev. Biol.
  • growth-promoting molecules can modify the effect of those molecules which normally function to inhibit neurite outgrowth and vice versa. Therefore, by varying the ratio of attractive or adhesive molecules to inhibitory molecules on or around glia or modifying their temporal appearance, a wide range of neurite patterns can be elicited. The range extend ⁇ from complete ⁇ eparation between the glial border and all adjacent axons (e.g. the roof plate, chick subplate, and dorsal optic stalk) , to partial separation (a pattern of intermittent cro ⁇ ing like that of the right lane of Figure
  • DS-PG Dermatan sulfate proteoglycan
  • KS/CS-PG keratan ⁇ ulfate/chondroitin ⁇ ulfate proteoglycan
  • Cellulose filter paper (Whatman #1) wa ⁇ cut into 350 ⁇ m strips and used to blot various proteoglycans onto the nitrocellulose substrate. The strips were soaked in 20 ⁇ l of the desired proteoglycan mixture. A solution of 1 mg/ml laminin (LN) was then ⁇ pread evenly acro ⁇ the dish with a bent glas ⁇ Pa ⁇ teur pipet. Quantitation of these procedures, and suitable controls, are described in detail in Section 7. , ⁇ upra.
  • Stripe ⁇ were made on the nitrocellulo ⁇ e-coated culture dishes with mixtures of LN (40 ⁇ g/ml) , and KS/CS- PG, or DS-PG at various concentrations.
  • PC-12 NEURON-LIKE CELL LINE PREPARATIONS The PC-12 cells used for the experiment were grown in media composed of DMEM plus 10% Horse Serum, 5% Fetal Calf Serum and 30 ⁇ g/ml gentamycin, final concentration. Confluent plates were disaggregated with 0.25% trypsin.
  • NGF Nerve Growth Factor
  • Stripes coated with protcogylcan which were completely inhibitory to neurite outgrowth were evaluated a ⁇ (-) , tho ⁇ e allowing slight outgrowth (+/-) , and those permis ⁇ ive to neurite outgrowth a ⁇ (+) .
  • PC-12 cells were plated on a substratum containing different concentrations of proteoglycans and grown in the presence of NGF. Neurite outgrowth was evaluated 24, 48 and 96 hours later, and is reported in
  • the growth cones of PC-12 neuron-like cells appear to share the same specificity for proteoglycan inhibition that DRG neurons demonstrate.
  • growth cones from both cell types may demonstrate a configurational specificity for C-4-S over C-6-S.
  • KS/CS-PG was shown to inhibit neurite outgrowth of dorsal root ganglia (DRG from chick E6, Section 7.2.1., supra) .
  • DRG dorsal root ganglia
  • DS-PG inhibition of neurite outgrowth was assayed.
  • DRGs Choick E 6 dorsal root ganglia
  • NGF Nerve Growth Factor
  • Stripes coated with DS-PG which were completely inhibitory to neurite outgrowth were evaluated as (-) , those allowing ⁇ light outgrowth (+/ ⁇ ) , and tho ⁇ e permissive to neurite outgrowth as (+) .
  • DRG dorsal root ganglia
  • DS-PG dermatan sulfate proteoglycan
  • DRG neurite outgrowth was completely inhibited by as little as 0.4 mg/ml (5 ⁇ M) DS-PG, and partly inhibited by 0.2 mg/ml (2.5 ⁇ M) DS-PG.
  • DRG cells are slightly more sensitive to inhibition by DS-PG than the neuron-like cell line PC-12. DRG cells are partly inhibited from outgrowth by as little as 0.2 mg/ml DS-PG, whereas PC-12 cell ⁇ were partly inhibited by 0.4 mg/ml DS- PG.
  • Dermatan sulfate proteoglycan (DS-PG) and keratan sulfate/chondroitin sulfate proteoglycan (KS/CS-PG) were found to inhibit glial cell and astrocyte invasion.
  • C-6 rat glial tumor cells and MCG-28 young immortalized mouse astrocytes were unable to invade the proteoglycan coated substratum for up to 96 hours.
  • Tis ⁇ ue culture Petri dishes 60-mm were coated with nitrocellulose (Schleicher & Schuell, Type BA 85 : 0.5 ml of a 5 cm 2 secti.on di.s ⁇ olved in 6 ml methanol) .
  • Cellulose filter paper (Whatman #1) was cut into 350 ⁇ m strips and used to blot various proteoglycans onto the nitrocellulose substrate. The strips were soaked in 20 ⁇ l of the desired proteoglycan mixture. A solution of 1 mg/ml laminin (LN) was then spread evenly across the dish with a bent gla ⁇ s Pasteur pipet. These methods are also described in Section 7.1.1, supra.
  • Stripes were made on the nitrocellulose-coated culture dishes with mixtures of LN (40 ⁇ g/ml) , and KS/CS-PG or DS-PG at various concentrations.
  • the different cell lines used for the experiment were grown in media composed of DMEM plus 5% Fetal Bovine Serum, 5% Calf Serum and 30 ⁇ g/ml gentamycin. Confluent plates were disaggregated with 0.25% trypsin. Plates used for experimental procedures were seeded at a ratio of 1:6 from confluent plates.
  • C-6 rat glial tumor cells (Paganetti et al, 1988, J. Cell Biol. 107:2291-2291) and MCG-28 young immortalized mouse astrocytes (a murine neonatal astrocyte line immortalized with SV-40) , were utilized for these experiments.
  • Stripes coated with proteoglycan which were completely inhibitory to cell invasion were evaluated as (-) , those allowing ⁇ light invasion (+/-) , and those permissive to invasion a ⁇ (+) ( Figure 26) .
  • C-6 glial cells were plated on different concentrations of DS-PG. Plate ⁇ were evaluated after 3, 24 and 48 hour ⁇ , and 5 days. The result ⁇ are shown in Table
  • MCG-28 immortalized young astrocyte ⁇ were plated on different concentrations of DS-PG. Plates were evaluated 24, 48, and 72 hours, and 5 days later. The results are shown in Table 4. TABLE 4. INHIBITION OF MCG-28 CELL INVASION ON DS-PG
  • DS-PG inhibited outgrowth of both cell lines comparably; no obvious enhancement of inhibition of glial cells (C-6) or astrocytes (MCG-28) was observed.
  • C-6 and MCG-28 cells were grown on variou ⁇ concentration ⁇ of KS/CS-PG, and evaluated at several time points later. The re ⁇ ults are shown in Table 5.
  • the different cell lines were grown on either KS/CS-PG or DS-PG coated stripe ⁇ . Plate ⁇ were evaluated for the extent of cell inva ⁇ ion after 24 and 48 hour ⁇ in culture. The results for growth on KS/CS-PG are shown in Table 6. The result ⁇ for growth on DS-PG are shown in Table 7. TABLE 6.
  • DISCUSSION DS-PG is a more potent inhibitor than KS/CS-PG when compared at equal dry weight concentration.
  • the KS/CS- and DS-PGs were found to inhibit outgrowth comparably.
  • proteoglycans may exert a fine regulatory action on the growth of neurons and non-neuronal cells in vivo, thus spacially regulating cell growth.

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THE JOURNAL OF NEUROSCIENCE, vol. 3, no. 11, November 1983, pages 2324-2335; S. CARBONETTO et al.: "Nerve fiber growth in culture on fibronectin, collagen, and glycosaminoglycan substrates" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0747470A1 (de) 1995-06-08 1996-12-11 The Procter & Gamble Company Keratanase enthaltende Reiningunszusammensetzungen

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