EP0821591A1 - Cns neurite outgrowth modulators, and compositions, cells and methods embodying and using same - Google Patents

Cns neurite outgrowth modulators, and compositions, cells and methods embodying and using same

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Publication number
EP0821591A1
EP0821591A1 EP96912910A EP96912910A EP0821591A1 EP 0821591 A1 EP0821591 A1 EP 0821591A1 EP 96912910 A EP96912910 A EP 96912910A EP 96912910 A EP96912910 A EP 96912910A EP 0821591 A1 EP0821591 A1 EP 0821591A1
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Prior art keywords
cells
neural
mammal
adhesion molecule
cam
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EP96912910A
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German (de)
English (en)
French (fr)
Inventor
Melitta Schachner
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Acorda Therapeutics Inc
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Acorda Therapeutics Inc
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • This invention relates generally to the modulation of neural growth in the central nervous system, and more particularly to methods and associated agents, constructs and compositions for improving CNS neural growth. Specifically, the invention relates to the use of cellular adhesion molecules, and preferably neural cell adhesion molecules such as L 1 , to foster and improve such neural growth.
  • Glial cells are the decisive determinants for controlling axon regrowth. Mammalian glial cells are generally permissive for neurite outgrowth in the central nervous system during development (Silver et al. (1982) J Comp. Neurol. 210:10-29; Miller et al. (1985) Develop. Biol. 111:35-41; Pollerberg et al. (1985) J. Cell. Biol. 101:1921-1929) and in the adult peripheral nervous system (Fawcett et al. (1990) Annu. Rev. Neurosci 13:43-60).
  • glial cells of the adult mammalian peripheral nervous system can revert to some extent to their earlier neurite outgrowth-promoting potential, allowing them to foster regeneration (Kalderon (1988) J. Neurosci Res. 21:501-512; Kliot et al. "Induced regeneration of dorsal root fibres into the adult mammalian spinal cord," In: Current Issues in Neural Regeneration, New York, pp. 31 1-328; Carlstedt et al. (1989) Brain Res. Bull. 22:93-102). Glial cells of the central nervous system of some lower vertebrates remain permissive for neurite regrowth in adulthood (Stuermer et al. (1992) J Neurobiol. 23:537-550). In contrast, glial cells of the central nervous system of adult mammals are not conducive to neurite regrowth following lesions.
  • Ll accumulates at sites of contact between neurons and Schwann cells beine concentrated mainlv at the cell surface of Schwann cells but not neurons (Martini et al. (1994a)). Furthermore, the homophilic binding ability of L 1 is enhanced by molecular association with the neural cell adhesion molecule N-CAM, allowing binding to occur through homophilic assistance (Kadmon et al. (1990a); Kadmon et al. (1990b) J. Cell Biol. 1 10:209-218 and 1 10: 193-208; Horstkorte et al. (1993) J Cell. Biol. 121 :1409- 1421).
  • Ll Besides its neurite outgrowth promoting properties, Ll also participates in cell adhesion (Rathjen et al. (1984) EMBO J. 3:1-10; Kadmon et al. (1990b) J. Cell. Biol. 110:209-218; Appel et al. (1993) J Neurosci., 13:4764-4775), granule cell migration (Lindner et al. (1983) Nature 305:427-430) and myelination of axons (Wood et al. (1990) J Neurosci 10:3635-3645).
  • L 1 consists of six immunoglobulin-like domains and five fibronectin type III homologous repeats.
  • Ll acts as a signal transducer, with the recognition process being a first step in a complex series of events leading to changes in steady state levels of intracellular messengers.
  • the latter include inositol phosphates, Ca 2 +, pH and cyclic nucleotides (Schuch et al. (1990) Neuron 3:13-20; von Bohlen und Hallbach et al. (1992) Eur. J. Neurosci. 4:896-909: Doherty et al. (1992) Curr. Opin. Neurobiol.
  • Ll is also associated with a casein type II kinase and another unidentified kinase which phosphorylates Ll (Sadoul et al. (1989) J Neurochem 328:251-254). Ll -mediated neurite outgrowth is sensitive to the blockage of L type Ca 2* channels and to pertussis toxin.
  • Ll Given the capability of Ll to promote neurite outgrowth, it is pertinent to investigate whether astrocytic expression of L 1 and other members of the immunoglobulin superfamily to which L 1 belongs, may overcome potentially inhibitory molecular cues reported to be present on glial cells and myelin in the adult central nervous system (Schachner et al., Perspectives in Developm.
  • an agent and corresponding methods are disclosed for the modulation of neural growth and particularly, such growth as can be promoted in the compartment of the central nervous system (CNS). and specifically, in myelinated nerve tissue.
  • the agents of the present invention are notable in their ability to promote such neural growth in an environment that has been traditionally viewed as inhibitory to the growth promoting stimulus of known neurite outgrowth factors. Specifically, this inhibitory environment includes inhibitory molecular cues which are present on glial cells and myelin the central nervous system.
  • the agents of the present invention are broadly selected from a group of cell adhesion molecules, and more preferably neural cell adhesion molecules. Most preferably, the agents of the present invention are selected from the group of molecules belonging to the immunoglobulin superfamily. and particularly to those members that mediate Ca 2 ⁇ -independent neuronal cell adhesion, of which L 1 , N- CAM and myelin-associated glycoprotein are particular members. Other cell adhesion molecules which may also influence CNS neural growth include laminin. fibronectin. N-cadherin, BSP-2 (mouse N-CAM). D-2, 224-1A6-A1, Ll-CAM. NILE. Nr-CAM. TAG-1 (axonin-1), Ng-CAM and F3/F1 1.
  • the agents of the invention belong to a new family referred to herein as the L 1 family of neural recognition molecules.
  • This family includes Ll, NgCAM, neurofascin, Drosophila neuroglian, zebrafish Ll. l and L1.2. and others.
  • This group of agents all demonstrate the Ig-like domains and FN-like repeats that are characteristic of L 1 , and in this connection, exhibit a remarkable colinearity, a high degree of N-glycosidically linked carbohydrates, which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kD band and smaller bands of 165 and 125 kD.
  • the agents of the present invention also include fragments of cell adhesion molecules and cognate molecules, congeners and mimics thereof which modulate neurite growth in the CNS.
  • the agents include molecules which contain structural motifs characteristic of extracellular matrix molecules, in particular the f ⁇ bronectin type III homologous repeats and immunoglobulin-like domains.
  • these structural motifs include those structurally similar to fibronectin type III homologous repeats 1-2, and immunoglobulin-like domains I-II, III-IV and V-VI.
  • the invention extends to methods of promoting and enhancing neural regeneration in vivo, and to the corresponding genetic constructs, such as plasmids. vectors, transgenes, and the like, and to pharmaceutical compositions, all of which may be used to accomplish the objectives of such methods.
  • the agents of the present invention may be prepared as vectors or plasmids, and introduced into neural cells located at a site in the CNS where regeneration is needed, for example, by gene therapy techniques, to cause the expression of an agent of the present invention and to thereby promote the requisite neural growth.
  • Another strategy contemplates the formulation of one or more of the appropriate agents in a composition that may likewise be directly delivered to a CNS site, as by parenteral admimstration.
  • the administration of such a composition may serve the purpose of inhibiting rather than promoting neural growth. This effect may be desirable in particular instances where unwanted or uncontrolled growth may occur or is occurring, and therefore the invention extends to this use as well.
  • the capability of the agents to engage in homophilic binding renders antagonists to the agents, including antibodies thereto, capable of acting as agonists, and thereby participating in the promotion of neural growth and regeneration.
  • the invention extends to the preparation of appropriate constructs and compositions containing the antibodies to the agents, for the therapeutic purposes set forth herein.
  • antibodies to Ll for example, may serve as part of a drug discovery assay or the like, to identify further agents that may possess activity and utility both diagnostic and therapeutic, in accordance with the present invention.
  • an L 1 analog antibodies such as polyclonal antibodies
  • the invention may be used to identify further members of the L 1 CAM family, and the invention accordingly extends to such CAM members as are isolated by use of such antibodies.
  • the invention also covers diagnostic applications, where for example, it is desirable to assess the potential for or actual development of CNS neural growth by the detection and measurement of the presence, amount or activity of one or more of the agents of the invention.
  • the invention also extends to assays, including drug discovery assays, that capitalize on the activity of the agents of the present invention in the modulation of CNS neural growth.
  • prospective drugs may be tested for CNS neural growth modulation by means of an assay containing an agent of the invention, or a cell line or culture developed in conjunction herewith may serve as the assay system.
  • the present invention also features transgenic mouse lines expressing a neural adhesion molecule in differentiated astrocytes and glial cells, and cells and tissues derived therefrom.
  • the neural adhesion molecule is Ll .
  • the invention features methods for enhancing neuronal outgrowth of CNS neurons, for enhancing memory and for increasing synaptic efficacy, as measured by stabilization of long term potentiation, and other similarly useful methods. Also featured are methods of testing drugs and other manipulations which modulate the effects of the neural adhesion molecule, and assay systems suitable for such methods.
  • a further object of the invention is to provide a cell culture containing the glial cells of the transgenic mammal.
  • Yet another object of the invention is to provide a cell culture system containing lesioned or unlesioned optic nerves or other parts of the nervous system of the transgenic mammal.
  • Still a further object of the invention is to provide a method for enhancing neuronal outgrowth of CNS neurons, which includes culturing the neurons on the glial cell culture system.
  • a further object of the invention is to provide a method for enhancing neuronal outgrowth of CNS neurons, which includes culturing the neurons on the optic nerve or other parts of the nervous system placed in the cell culture system.
  • a still further object of the invention is to provide a method for enhancing neuronal outgrowth of CNS neurons, which includes the secretion of neural adhesion molecule by implanted cells.
  • Another object of the invention is to provide a method for enhancing memory, which includes administering to the brain of a mammal in need of memory enhancement, an amount of the cells of the glial cell culture system effective to enhance the memory of the mammal.
  • Yet another object of the invention is to provide a method for enhancing memory, including administering to the brain of a mammal in need of memory enhancement, an amount of the cells of the optic nerve or other parts of the nervous system placed in the cell culture system effective to enhance the memory of the mammal.
  • a still further object of the invention is to provide a method for enhancing memory, including delivering to the glial cells of the brain of a mammal in need of such memory enhancement, a vector which allows for the expression of a neural adhesion molecule in the glial cells.
  • a further object of the invention is to provide a method for enhancing memory, which includes the secretion of neural adhesion molecule by implanted cells.
  • Another object of the invention is to provide a method for increasing synaptic efficacy in the CNS of a mammal in need of such an increase, including administering to the brain of the mammal, an amount of the cells of the glial cell culture system effective to increase synaptic efficacy in the brain of the mammal.
  • Yet a further object of the invention is to provide a method for increasing synaptic efficacy in the CNS of a mammal in need of such an increase, including administering to the brain of the mammal, an amount of the cells of the optic nerve or other parts of the nervous system placed in the cell culture system effective to increase synaptic efficacy in the brain of the mammal.
  • a still further object is to provide a method for increasing synaptic efficacy in the CNS of a mammal in need of such an increase, which includes delivering to the glial cells of the brain of a mammal in need of such enhancement, a vector which allows for the expression of a neural adhesion molecule in the glial cells.
  • a further object of the invention is to provide a method for increasing synaptic efficacy, which includes the secretion of neural adhesion molecule by implanted cells.
  • Another object of the invention is to provide a method of testing the ability of a drug or other entity to modulate the activity of a neural adhesion molecule, which includes adding CNS neurons to the glial cell culture system; adding the drug under test to the cell culture system; measuring the neuronal outgrowth of the CNS neurons; and correlating a difference in the level of neuronal outgrowth of cells in the presence of the drug relative to a control culture to which no drug is added to the ability of the drug to modulate the activity of the neural adhesion molecule.
  • Another object of the invention is to provide a method of testing the ability of a drug or other entity to modulate the activity of a neural adhesion molecule which includes adding CNS neurons to the optic nerve or other parts of the nervous system cell culture system; adding the drug under test to the cell culture system; measuring the neuronal outgrowth of the CNS neurons; and correlating a difference in the level of neuronal outgrowth of cells in the presence of the drug relative to a control culture to which no drug is added to the ability of the drug to modulate the activity of the neural adhesion molecule.
  • Yet another object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes the glial cell culture system; and CNS neurons added to the cell culture system.
  • a further object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes culturing the glial cell culture system inoculated with a drug or agent; adding CNS neurons to the cell culture system; and examining neuronal outgrowth to determine the effect of the drug thereon.
  • Yet another object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes culturing the optic nerve or other parts of the nervous system in the cell culture system inoculated with a drug or agent; adding CNS neurons to the cell culture system; and examining neuronal outgrowth to determine the effect of the drug thereon.
  • Another object of the invention is to provide an assay system for screening drugs and other agents for ability to modulate the production of a neural adhesion molecule, which includes inoculating a culture of CNS neurons with a drug or agent: adding a soluble neural adhesion molecule: and examining neuronal outgrowth to determine the effect of the drug thereon.
  • FIGURE 1 depicts the map of the GFAP-L1 chimeric transgene.
  • a 4.05 kb mouse Ll cDNA was inserted into exon 1 of a modified GFAP gene using Not I linkers.
  • the Ll cDNA is preceded 5' by an SV40 late gene splice (V) and followed 3' by an SV40 polyadenylation signal (pA).
  • V SV40 late gene splice
  • pA polyadenylation signal
  • the locations of the L l ATG and the polyadenylation signal are indicated. Exons are shown as boxes.
  • FIGURE 2 depicts a Northern blot analysis of brain RNA from different transgenic lines. 10 ⁇ g of total RNA of whole adult brain was loaded in each lane and probed with mouse L l cDNA. Exposure time was 3 days. Lanes 1-3, brains from different transgenic offspring (lane I : line 3426; lane 2: line 3427; lane 3: line 3418; lane 4, brain from non-transgenic control). Note that the level of transgenic L 1 mRNA (arrow) is different in the three transgenic lines, with levels being highest in line 3426, intermediate in line 3427 and lowest in line 3418. The position of 28 S and 18S rRNA is shown on the right margin.
  • FIGURE 3 depicts the localization of L 1 mRNA in adult unlesioned (A, C and E) and lesioned (15 days after the lesion.
  • B and D optic nerves from non-transgenic (A, B and E) and transgenic mice (C and D) of line 3426 by in situ hybridization.
  • L 1 mRNA is detectable only in neuronal cells of the retina but not in the glial cells of the optic nerve (A and B).
  • transgenic animals cells containing Ll transcripts are visible in the optic nerve (C and D). The density of Ll positive cells is highest in the unmyelinated proximal part of the nerve.
  • the density of L 1 mRNA positive cells in the nerve is slightly increased after a lesion (compare C and D).
  • the distribution of cells expressing Ll (C and D) is similar to that of cells expressing GFAP (E).
  • Scale bar in E 100 ⁇ m (for A to E).
  • FIGURE 4 depicts the double immunofluorescence microscopic localization of L 1 (A and B) and GFAP (C) in unlesioned (A and C) and lesioned (28 days after the lesion, B) optic nerves from adult transgenic (line 3426, A and B) and wild type (C) animals.
  • Ll immunoreactivity in optic nerves from transgenic animals is significantly increased after a lesion (compare A and B).
  • the pattern of Ll immunoreactivity in lesioned transgenic nerves is similar to the pattern of GFAP immunostaining in unlesioned wild type nerves.
  • Ll positive unmyelinated retinal cell ganglion axons are present in unlesioned wild type nerve (A). Scale bar in C:50 ⁇ m (For A to C).
  • FIGURE 5 depicts the double immunofluorescence microscopic localization of L 1 (A and D) and GFAP (B and E) in cultured astrocytes from transgenic animals of 6/3295
  • FIGURE 6 shows (A) Western blot analysis of lesioned (15 days after the lesion) and unlesioned optic nerves from transgenic and wild type animals. 25 ⁇ g of total protein of lesioned (lanes 1. 3 and 5) or unlesioned nerves (lanes 2. 4, and 6) was loaded and detected using affinity purified polyclonal antibodies against L 1. Protein extracts were made from mice of transgenic lines 3426 (lanes 1 and 2), 3427 (lanes 3 and 4) and from wild type animals (lanes 5 and 6). There is an increase in L 1 expression in transgenic animals compared to non-transgenic controls. Following optic nerve lesion, an up-regulation of L 1 occurred in transgenic animals, whereas the amount of L 1 in wild type animals decreased. Apparent molecular weights (in kD) are shown on the left margin.
  • FIGURE 7 depicts examples of neurite outgrowth from mouse cerebellar neurons cultured on cryostat sections of optic nerves from wild type (A and B) and transgenic (C and D) animals (line 3426).
  • a and C represent unlesioned optic nerves
  • B and D represent lesioned optic nerves.
  • FIGURE 8 depicts and compares neurite lengths of cerebellar neurons maintained on cryostat sections of unlesioned (c) and lesioned (1) optic nerves (28 days after the lesion) from wild type (WT) and transgenic animals (lines 3426, 3427 and 3418). Note that the length of neurites on sections from transgenic animals is greater than on sections from wild type animals. In transgenic lines neurites are always longer on lesioned than on unlesioned nerves, whereas neurite lengths on unlesioned and lesioned nerves of wild type animals do not show a significant difference. Note that the neurite length correlates positively with the levels of Ll expression in different transgenic lines (see also Western blot data). Mean values ⁇ 13 standard error of the mean from one representative experiment (out of 12) are shown.
  • FIGURE 9 is a graph measuring neurite lengths of cerebellar neurons maintained on cryostat sections of unlesioned (c) and lesioned (1) optic nerves (28 days after the lesion) from wild type (WT) and transgenic animals without and after pre ⁇ incubation of sections with affinity purified polyclonal antibodies against Ll (anti Ll) and mouse liver membranes (anti liver). Neurite lengths on nerves without pre-incubation with any antibody were taken as 100% and neurite lengths on sections of the same nerves obtained after antibody treatment were expressed in relation to this value. A significant reduction (60%) of neurite length by L 1 antibodies was found on cryostat sections from transgenic animals. Numbers on the top represent the total number of nerves measured for each value. Mean values
  • ⁇ standard error of the mean are from at least four independent experiments carried out in duplicate.
  • FIGURE 10 depicts and compares neurite lengths of mouse cerebellar (A) or chick DRG (B) neurons on astrocytic monolayers prepared from wild type (WT) and transgenic animals (line 3426) in the absence of antibodies and after pre-incubation of sections with affinity purified polyclonal antibodies against L2 (anti L 1 ) and mouse liver membranes (anti liver).
  • the neurite length on astrocytes without pre- incubation with any antibody was taken as 100% and the neurite lengths on astrocyte monolayers obtained after antibody treatment are expressed in relation to this value.
  • a significant reduction (about 40%) of neurite length is only visible on transgenic astrocytes after preincubation of the monolayers with L 1 antibodies.
  • Mean values ⁇ standard deviation are from at least 100 neurons from two independent experiments carried out in quadruplicate. * indicates means that were significantly different (p ⁇ 0.05, Mann- Whitney U test) from the control (wild type or transgenic astrocytes without any antibody treatment).
  • FIGURE 1 1 demonstrates the in vivo regrowth of axons in the optic nerve (0-2000 ⁇ m). 6-8 week old GFAP-L1 transgenic mice and wild type mice were crushed folklor, ⁇ ft , origin, n
  • FIGURE 12 depicts in vivo regrowth of axons in the optic nerve (0-800 ⁇ m). 6-8 5 week old GFAP-L 1 transgenic mice and wild type mice were crushed intraorbitallv and, after 14 days, traced with a fluorescein-labeled biotin ester to mark retinal ganglion cell axons by anterograde labeling. Each point represents one animal.
  • FIGURE 13 shows the effect of the injection of chicken Ll antibodies into the IMHV on percent avoidance (retention of memory) on a one-trial passive avoidance 10 task.
  • Each point represents a group of birds who received injections of Ll antibodies (anti-L 1 ) (closed circles) or saline (open squares) at the time relative to training indicated. All animals were tested at 24 hours post-training (*, p ⁇ 0.05 between saline and antibody groups, ⁇ 2 ).
  • FIGURE 14 comprises two graphs depicting the effect of injections of Ig I-IV and 15 FN fragments at -30 minutes and +5.5 hours on retention of memory for passive avoidance task. All animals were tested at 24 hours post-training. The number of animals in each group is shown in the histograms (*p ⁇ 0.05; **p ⁇ 0.005).
  • FIGURE 15 comprises a series of graphs showing the influence of antibodies against Ll (anti-L 1) on LTP in pyramidal neurons in the CA1 region of rat
  • FIGURE 16 demonstrates the influence of the immunoglobulin-like domains I-VI. polyclonal NCAM antibodies and oligomannosidic glycopeptides on LTP. a, time- course of the EPSP initial slope before and after TBS in the presence of the immunoglobulin (Ig)-like domains I-VI (216 ⁇ g/ml; 3.2 mM; in 20 mM Tris HCl pH 7.6 O; p ⁇ 0.01) and the fibronectin (FN) type III homologous repeats I-V (225 ⁇ g/ml; 3.8 mM; in 20 mM Tris/HCl pH 7.6, ⁇ ) of Ll, compared to control LTP (20 mM Tris/HCl, pH 7.6, D).
  • Ig immunoglobulin
  • FN fibronectin
  • TBS in the presence of oligomannosidic glycopeptides (O).
  • FIGURE 17 graphically depicts the influence of Ll antibodies and oligomannosidic carbohydrates on previously established LTP and on MNDA receptor-mediated synaptic transmission, a, Time-course of the EPSP initial slope before and after TBS in the presence of Ll antibodies applied either throughout the experiment (6.2
  • FIGURE 18 depicts the nucleotide sequence of the 4.43 kb cDNA insert of clone pX#2 and deduced amino acid sequence of mouse CHL 1.
  • the longest open reading frame (bp 296 to bp 3922) contains 1209 amino acids terminating with a TGA termination codon.
  • the two hydrophobic regions representing the signal peptide (amino acids 1-24) and the transmembrane region (1082-1 104) are
  • F3; W 830, F4: W 936, F5; W 1053) and tyrosines/phenylalanines are boxed.
  • a bracket highlights the RGD and DGEA sequences (amino acid residues 185-187 and 555-558, respectively). Untranslated sequences are shown numbered in italics. The sequence data are available from EMBL/Genebank/DDBJ under accession number X94310.
  • FIGURE 19 depicts the domain structure, coding region of the bacterially expressed protein fragment, and hydrophobicity plot of mouse CHL 1 (a)
  • the diagram sketches the structural features deduced from the primary sequence of CHL 1. Numbers refer to the amino acid sequence starting at the translation start site, lg-like domains I to VI are represented by half circles (amino acid numbers refer to the cysteines forming the disulfide bridges). FN-like repeats 1 to 5 are symbolized by boxes (amino acid numbers refer to the domain boundaries). The potential sites for N-glycosylation are indicated by filled circles.
  • Signal peptide and transmembrane region are denoted by etched boxes, (b) The bar indicates the position of the cDNA encoding the recombinant protein produced in E. coli (c) The hydrophobicity plot (Kyte and Doolittie, 1982) of the deduced amino acid sequence shows the characteristic features of an integral membrane protein with the putative hydrophobic signal sequence and transmembrane domain (*). Positive values indicate hydrophobicity. Numbering of the abscissa refers to amino acid position.
  • FIGURE 20 shows the alignment of the intracellular domains of molecules of the Ll family.
  • the sequences of the intracellular domains starting with the first amino acid residue after the putative transmembrane regions are aligned for mouse CHLl , mouse Ll. chicken Nr-CAM. chicken Ng-CAM, chicken neurofascin. Drosophila neuroglia. and zebrafish L1.2.
  • the numbers refer to the amino acid positions of CHLl and gray boxes indicate gaps introduced in the CHLl sequence. Identical amino acids occurring in the majority of sequences are marked by black boxes.
  • the three brackets (I. II and III) refer to highly conserved stretches.
  • FIGURE 21 is a Northern blot analysis of CHLl and Ll mRNA in different tissues of mouse and rat
  • Poly A
  • RNA markers are indicated at the left margins.
  • FIGURE 22 demonstrates the specificity of polyclonal antibodies against CHL 1 and expression of CHL 1 in different tissues.
  • FIGURE 23 shows the detection of CHL 1 on transiently transfected COS- 1 cells
  • Monolayer cultures of CHL 1 -transfected (a) and mock-transfected (c) COS- 1 cells were immunostained with polyclonal antibodies against CHL 1.
  • (b,d) corresponding phase contrast micrographs for (a.c). respectively. Bar in d 30 ⁇ m for a to d.
  • FIGURE 24 depicts the localization of CHLl and Ll mRNA in sections of mouse retina, optic nerve, and cerebellar cortex by in situ hybridization analysis.
  • L 1 mRNA is detectable in ganglion cells located in the ganglion cell layer (1 in a) and in amacrine and horizontal cells located in the inner nuclear layer (2 in 1 ).
  • Other cells types in the retina or glial cells in the optic nerve do not contain detectable levels of Ll transcripts (a).
  • CHLl mRNA is weekly detectable in ganglion cells and in a few cells located at the inner (i.e. vitread) margin of the inner nuclear layer (b).
  • FIGURE 25 illustrates the immunofluorescence microscopic localization of CHL 1 in cultures of astrocytes.
  • FIGURE 26 is a Western blot analysis of deglycosylated CHL 1 Soluble (S) and insoluble (M) fractions of detergent lysates of crude membranes from brain of seven-day-old mice were incubated with N-glycosidase F (N). O- glycosidase (O), both enzymes (N+O). or without enzyme (-) and reacted with antibodies against CHL 1 in Western blots. Molecular mass standards are indicated in kD at the right margin. The molecular masses of the glycosylated and deglycosylated CHL 1 protein components are indicated in kD in the box below.
  • FIGURE 27 shows the presence of the MNK-1 carbohydrate in CHLl immimoprecipitates from brain tissue.
  • CHL 1 was immunoprecipitated from detergent lysates of whole brain tissue of nine-day-old mouse brain using CHL 1 antibodies.
  • Brain lysate (lane 1) and immunoprecipitates (lanes 2,3) were resolved by SDS-PAGE, blotted, and incubated with monoclonal antibody 312 against the HNK-1 epitope (lanes 1,2) or CHLl antibodies (lane 3).
  • Molecular mass standards are indicated in kD at the right margin.
  • FIGURE 28 NEURITE OUTGROWTH OF HIPPOCAMPAL NEURONS IN COCULTURES WITH L929-TRANSFECTANTS
  • Hippocampal neurons derived from rats of embryonal day 18 were cultured in subconfluent monolayers of L929-transfectants or parental L929 cells. After 1 1-12 h of coculture the cells were fixed and labeled with monoclonal antibody412
  • CHL 1 -transfectants CHLl
  • L929 parental L929 cells
  • (+AB polyclonal antibodies against recombinant CHLl
  • C Neurite outgrowth promotion affects all length classes of neurites. Cumulative frequency distribution plot of the total neurite length of hippocampal neurons cocultured with CHL 1 -transfectants (CHLl line 1 and 2) and parental L929 cells (L929) with (+AB) or without antibody treatment as given in (A). The percentage of neurons with neurites longer than or equal to a certain length x (vertical axis) was plotted as a function of neurite length x (horizontal axis). Values are from one representative experiment.
  • FIGURE 29 NEURITE OUTGROWTH OF SMALL CEREBELLAR NEURONS IN COCULTURE WITH L929-TRANSFECTANTS Cerebellar neurons derived from 6-7 day old mice were cultured for 20 h on
  • CHLl CHLl -transfectants
  • Mock CHL 1 -transfected non-expressing L929 cells
  • parental L929 parental L929
  • Ll L 1 -transfectants
  • CHL 1 promotes neurite outgrowth of small cerebellar neurons. The mean of total neurite length of three experiments is shown. Error bars are standard error of the mean.
  • CHLl promotes neurite outgrowth also of small cerebellar neurons better than Ll .
  • the total neurite length is given as percent of L929 cells as a control (ctr). Error bars are standard error of the mean.
  • C Increase of neurite outgrowth of cerebellar neurons by CHL 1 affects all size classes of neurites. Cumulative frequency of distribution plot of the total neurite length of the percentage of neurons with neurites longer than or equal to a certain length x (vertical axis) was plotted as a function of neurite length x (horizontal axis). Values from one representative experiment are shown. 6/32959
  • FIGURE 30 NEURITE OUTGROWTH OF HIPPOCAMPAL NEURONS TREATED WITH SOLUBLE CHLl
  • Hippocampal neurons were cultured on poly-L-lysine coated coverslips for 12 h with addition of supernatants (40 ⁇ g/ml of total protein) of crude membrane preparations of CHL 1 -transfectants (CHLl), parental L929 cells (L929), or Ll- transfectants (Ll). Staining and measurement of neurite length was performed as already described ( Figure 7).
  • Soluble CHL 1 promotes a slight increase of neurite number.
  • Total neurite length in percent of the neurite length of hippocampal neurons treated with supernatants derived from parental L929 cells (ctr) are plotted. Values are means of three independent experiments. Error bars are standard error of the mean.
  • C Also soluble CHL 1 affects neurite outgrowths of all length classes of neurites. Cumulative frequency distribution plots of the total neurite length from one representative experiment are shown.
  • FIGURE 31 QUANTITATIVE AGGREGATION ANALYSIS AND STABILITY OF CHL 1 - AND L 1 -PROTEIN IN L929 TRANSFECTANTS.
  • CHLl- (CHLl) (ctr) and Ll- (Ll) transfected cells were cultured (at densities of about 3xl0 6 cells/ml) for 18 h in culture medium with (+ind) or without (-ind) induction of transgene expression by CuSO .
  • Particle number was counted in a hemacvtometer at the beginning and at the end of the incubation. The percentage of aggregation was calculated by the index (l-N/NO)xl00.
  • N18 and NO represent the particle numbers at the end or the beginning of the incubation period, respectively. Values are the means of at least four independent experiments. Error bars are standard deviations. 6/329
  • the present invention relates to the use of certain agents identified herein as "CNS neural growth modulators” (CNGMs), and particularly to a class of neural cell adhesion molecules as defined herein, to promote neurite outgrowth in the central nervous system (CNS).
  • CNGMs central nervous system
  • neurons in the adult central nervous system have been considered incapable of regrowth, due to inhibitory molecular cues present on glial cells.
  • the agents and methods of the present invention can be used to overcome this inhibition and promote CNS neurite outgrowth.
  • the agents of the invention include and may be selected from any cell adhesion molecule which is capable of modulating or promoting CNS neurite outgrowth, and particularly to molecules belonging to the immunoglobulin superfamily. More particularly, the molecules are selected from the members of the immunoglobulin superfamily which mediate Ca 2* -independent neuronal cell adhesion, including L 1 , N-CAM and myelin-associated glycoprotein.
  • the invention also contemplates fragments of these molecules, and analogs, cognates, congeners and mimics of these molecules which have neurite-promoting activity.
  • Particularly preferable structural motifs for these fragments and analogs include domains similar to the fibronectin type III homologous repeats (particularly repeats 1-2) and immunoglobulin-like domains (particularly domains I-II. III-IV and V-VI). 6/32959
  • both the agents and their antagonists, and particularly, their antibodies may serve as agonists with respect to the receptor for the agents, and may thus be employed in both diagnostic and therapeutic applications in the same manner and for the same purpose as the agents themselves.
  • L 1 acts as a receptor, and its antibody may be employed as an agonist, to promote neurite outgrowth as set forth herein, to assist in neural regeneration particularly in the CNS.
  • the class of materials identified as CNS neural growth modulators hereinbelow is considered to include the antibodies to CAMs such as Ll and its analogs, such as CHL 1. described later on herein, among its numbers.
  • the present invention relates in one aspect to the ectopic expression of CNS neural growth modulators (CNGMs) or neural cell adhesion molecules on differentiated astrocytes in vivo.
  • CNGMs CNS neural growth modulators
  • These molecules have been found to enhance neurite outgrowth on monolayer cultures of such astrocytes and cryostat sections of unlesioned and lesioned adult mouse optic nerves, and also in vivo, in optic nerve crush experiments in transgenic animals.
  • the increased neurite outgrowth-promoting capacity is proportional to the level of ectopic CNGM expression. This is demonstrated by comparisons of the distinct transgenic lines of the invention, which express different basal levels of transgenic-encoded CNGM. and by correlations following increased CNGM expression after a lesion of the optic nerve.
  • any part of the nervous system can likewise be used, including portions of the brain and spinal cord.
  • Neurite outgrowth is dependent on the levels of CNGM expression by astrocytes, demonstrating the specific effect exerted by CNGM in promoting neurite outgrowth in the transgenic animal. Inhibition of neurite outgrowth by polyclonal CNGM antibodies, but not by antibodies to mouse liver membranes, further supports this specificity, in particular, since both antibodies react well with the cell surfaces of neurons and astrocytes of transgenic animals.
  • the CNGM is Ll.
  • L i ' s biological effects can be inhibited by Ll antibodies, which indicates that L l is homophilically active in a trans configuration at the cell surface of transgenic astrocytes.
  • Ll species-specific antibodies that do not react with chicken dorsal root ganglion neurons inhibit neurite outgrowth of this neuronal cell type on transgenic astrocytes.
  • the transgene-mediated enhancement of neurite outgrowth on glial cells that do not normally express L 1 in vivo indicates that glial cells of the adult mammalian central nervous system can be made more conducive to neurite outgrowth.
  • the loss of neurite outgrowth-promoting glia-derived molecules with maturation Smith et al. (1986) J. Comp. Neurol. 251 :23-43; Smith et al. (1990) £>ev. Biol. 138:377- 390
  • a recognition molecule that is normally highly expressed by glial cells in the adult mammalian peripheral nervous system (Niecke et al. (1985); Bixby et al. (1988) J. Cell. Biol. 107:353-362; Seilheimer et al. (1988) J Cell. Biol. 107:341-351).
  • the phenotype of adult astrocytes from the present transgenic lines may be modified towards the more Schwann cell-related capacity of reexpressing Ll after infliction of a lesion.
  • An increase in L l expression by Schwann cells is likely mediated by neurotrophins upregulated after damage by autocrine mechanisms (Seilheimer et al. (1987) EMBO J. 6: 1611-1616).
  • Ll expression by astrocytes in culture can be upregulated by TGF- ⁇ and NGF (Saad et al. (1991)).
  • L 1 may be particularly beneficial for neurite outgrowth in myelinated tracts of the central nervous system which normally contain several molecules that are neurite outgrowth inhibiting (Schachner et al.. Perspectives in Developm. Neurobiol. in Press; Schwab et al. (1995) Ann. Rev. Neurosci. 16:565-595).
  • the present invention demonstrates that the inhibitory action of astroglial and oligodendroglial cells may be overcome, at least in part, by the neurite outgrowth promoting properties of the agents defined herein, and as particularly illustrated by the activity of ectopically expressed L 1.
  • Expression of L 1 by astrocytes seems also to compensate for inhibitory effects exerted by oligodendrocytes.
  • Permissive and non-permissive molecular cues therefore may not have to be localized on the same cell type for neurite outgrowth to occur. Instead, such molecular cues might be partitioned among different cell types.
  • the cellular and molecular manipulation of L 1 and other neurite outgrowth promoting molecules may therefore allow enhancement of the regenerative capacity of the adult mammalian central nervous system following injury or disease.
  • the present invention extends to the promotion of neural growth in the CNS, including such growth as is desired to regenerate structures lost due to injury or illness, as well as those structures and tissues exhibiting incomplete or immature formation.
  • the agents of the invention also exhibit a neuroprotective or neuropreservative effect as illustrated later on herein, and for example, could be administered to inhibit or counteract neural degeneration or loss of variable etiology.
  • the invention accordingly extends to constructs and compositions containing or delivering the agents of present invention, whether by the promotion of the 27 expression of certain agents via gene therapy or the like, or by the exogenous admimstration of the agents where appropriate and beneficial, in pharmaceutical compositions to treat injured or diseased CNS structures.
  • certain of the agents are able to exert a growth promoting effect when so administered, although it is recognized that members of the presently identified group, such as Ll and N-CAM appear to bind homophilically and may therefore prove more beneficial when delivered by means of expression.
  • the invention is intended to extend to both routes and protocols where feasible.
  • the present invention relates to the use of CNGM- secreting cells for the modulation of neural outgrowth, regeneration, and neural survival in the CNS.
  • certain soluble CNGMs and fragments thereof, and cognate molecules thereof are also within the invention.
  • agent CNS neural growth modulator
  • CNGM nerve growth modulator
  • nerve recognition molecule recognition factor
  • recognition factor protein(s) recognition factor protein(s)
  • CN adhesion molecule any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence previously described and the profile of activities set forth herein and in the Claims.
  • the foregoing terms also include active fragments of such proteins, cognates, congeners, mimics and analogs, including small molecules that behave similarly to said agents.
  • proteins displaying substantially equivalent or altered activity are likewise contemplated.
  • modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis. or may be accidental, such as those obtained through mutations in hosts that are producers of the complex /32959
  • CNS neural growth modulator CNGM
  • CNGM CNGM
  • CN recognition factor recognition factor
  • recognition factor protein(s) recognition factor protein(s)
  • neural adhesion molecule e.g., aural adhesion molecule
  • amino acid residues described herein are preferred to be in the "L" isomeric form.
  • residues in the "D” isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property of immunoglobulin- binding is retained by the polypeptide.
  • NH refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • a “replicon” is any genetic element (e.g., plasmid. chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a "vector” is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine. guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids. and chromosomes.
  • linear DNA molecules e.g., restriction fragments
  • viruses e.g., plasmids. and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e.. the strand having a sequence homologous to the mRNA).
  • An "origin of replication” refers to those DNA sequences that participate in DNA synthesis.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g.. mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5 " direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to probes, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides. although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See. e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g.. a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • an “antibody” is any immunoglobulin. including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816.397 and 4.816,567.
  • an "antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • antibody molecule in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially 5 intact immunoglobulin molecules and those portions of an immunoglobulin .
  • molecule that contain the paratope including those portions known in the art as Fab, Fab', F(ab'), and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') portions of antibody molecules are prepared by the proteolytic 10 reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous et al. Fab * antibody molecule portions are also well- known and are produced from F(ab'), portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and 15 followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of 20 immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an 35 allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5x SSC and 65°C for both hybridization and wash.
  • the present invention relates to transgenic animals which express a CNGM or neural recognition molecule, in particular L 1 , and preferably in astrocytes. These animals have increased capability for neural outgrowth in the central nervous system.
  • the invention also includes an assay system for the screening of potential drugs effective to modulate neural outgrowth of target mammalian cells by interrupting or potentiating the CNGM's neural recognition activity.
  • neural recognition activity or “neural adhesion activity” is meant any biological effect which is a result of the CNGM ' s binding to another molecule, including intracellular effects on second messengers.
  • the test drug could be administered either to a cellular sample with the ligand that activates the CNS neural growth modulator, or a transgenic animal expressing the CNS neural growth modulator, to determine its effect upon the binding activity of the modulator to any chemical sample, or to the test drug, by comparison with a control.
  • Identifying characteristics of at least one of the present CNS neural growth modulators, in particular L 1 is its participation in changes in steady state levels of intracellular messengers, including Ca 2* , pH, and cyclic nucleotides, as well as changes in the activities of protein kinases such as protein kinase C, pp60 c " src . a casein type II kinase and another kinase known to phosphorylate L 1.
  • the assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the CNGMs or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating transcriptional activity.
  • Such assay would be useful in the development of drugs that would be specific to particular cellular activity, such as neural outgrowth or increase in synaptic efficacy, or that would potentiate such activity, in time or in level of activity.
  • drugs might be used to modulate neural outgrowth in response to injury, or to treat other pathologies, as for example, in treating neurodegenerative diseases such as Parkinson's Disease, ALS, Huntington's Disease and Alzheimer's Disease.
  • the invention contemplates agonists and antagonists of the activity of a CNS neural growth modulator.
  • an agent or molecule that inhibits the ability of neurons to recognize a CNGM such as L 1 can be used to block neural outgrowth, where such outgrowth is contraindicated, and as described earlier, a pharmaceutical composition containing such an agent may be administered directly to the target site.
  • an agonist can be a peptide having the sequence of a portion of an L 1 domain particularly that between fibronectin type III homologous repeats 2 and 3, or an antibody to that region.
  • Ll has the ability to undergo homophilic binding (i.e., Ll can bind to itself, and therefore both antibodies to Ll and fragments of Ll itself are capable of binding to Ll).
  • One of the diagnostic utilities of the present invention extends to the use of the present CNGMs in assays to screen for protein kinase inhibitors. Because the activity of the CNGMs described herein are phosphorylated, they can and presumably are dephosphorylated by specific phosphatases. Blocking of the specific kinase or phosphatase is therefore an avenue of pharmacological intervention that would modulate the activity of these neural recognition proteins.
  • the present invention likewise extends to the development of antibodies against the CNGMs, including naturally raised and recombinantly prepared antibodies.
  • the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the CNGMs.
  • Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating neural outgrowth.
  • antibodies against CNS neural growth modulators can be selected and are included within the scope of the present invention for their particular ability in binding to the protein.
  • activity of the neural growth modulators or of the specific polypeptides believed to be causally connected thereto may therefore be followed directly by the assay techniques discussed later on. through the use of an appropriately labeled quantity of the neural growth modulator or antibodies or analogs thereof.
  • the CNGMs. their analogs, and any antagonists or antibodies that may be raised thereto are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay. using for example, an antibody to the CNGM that has been labeled by either radioactive addition, reduction with sodium borohydride. or radioiodination.
  • a control quantity of the antagonists or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample. After the labeled material or its binding partner(s) has had an opportunity to react with sites within the sample, the resulting mass may be examined by known techniques, which may vary with the nature of the label attached. For example, antibodies against the CNGMs may be selected and appropriately employed in the exemplary assay protocol, for the purpose of following protein material as described above.
  • radioactive label such as the isotopes 3 H. I- C, 32 P, 3 S, 36 C1, 5l Cr, "Co, 58 Co, 59 Fe. "Y, 125 I, ,3I I, and l86 Re
  • known currently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric. fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence of the neural growth modulators, or to identify drugs or other agents that may mimic or block their activity.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the neural growth modulators, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand. capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the CNS neural growth modulator(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity.
  • a first therapeutic method is associated with the promotion of CNS neural growth resulting from the presence and activity of the CNGM, its active fragments, analogs, cognates, congeners or mimics, and comprises administering an agent capable of modulating the production and or activity of the CNGM, in an amount effective to promote CNS development, regrowth or rehabilitation in the host.
  • drugs or other neutralizing binding partners to the CNGM or proteins may be administered to inhibit or prevent undesired neural outgrowth.
  • the modulation of the action of specific kinases and phosphatases involved in the phosphorylation and dephosphorylation of CNGMs or proteins presents a method for modulating the activity of the modulator or protein that would concomitantly potentiate therapies based on CNGM/protein activation.
  • the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the admimstration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of the activity of the CNS neural growth modulator or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • pharmaceutical compositions may comprise effective inhibitors or enhancers of the activity of the CNS neural growth modulator or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • drugs or other binding partners to the CNS neural growth modulator or proteins may be administered to inhibit or potentiate binding and second messenger activity.
  • CHL 1 comprises an N-terminal signal sequence, six immunoglobulin (Ig)-like domains, and 4.5 fibronectin type III (FN)-like repeats, a transmembrane domain, and a C-terminal, most likely intracellular domain of approximately 100 amino acids.
  • CHLl is most similar in its extracellular domain to chicken Ng-CAM (about 40% amino acid identity), followed by mouse Ll, chicken neurofascin, chicken Nr-CAM, Drosophila neuroglian, and zebrafish Ll. l (37 to 28 % amino acid identity, respectively), and mouse F3, rat TAG-1.
  • the similarity with other members of the Ig superfamily is 16 to 1 1 %.
  • the intracellular domain is most similar to mouse and chicken Nr-CAM, mouse and rat neurofascin (about 50 % amino acid identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian, and zebrafish Ll . l and L1.2 (about 40 % amino acid identity).
  • Ll characteristic criteria were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig-like domains and FN-like repeats.
  • CHLl shared between members of the Ll family are a high degree of N-glucosidically linked carbohydrates (about 20% of its molecular mass), which include the HNK-1 carbohydrate structure, and a pattern of protein fragments comprising a major 185 kD band and smaller fragments of 100 and 125 kD.
  • HNK-1 carbohydrate structure As for the outer Ll family members, predominant expression of CHLl is observed in the nervous system and at later developmental stages. 41
  • the following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
  • Glial fibrillary acidic protein (GFAP. Eng et al. (1971) Brain Res. 28:351-354) is expressed predominately by astrocytes at late stages in the development of the mouse central nervous system (Landry et al. (1990) J Neurosci. Res. 25:194-203). Therefore regulatory sequences of the GFAP gene were used to direct the
  • the GFAP-L 1 transgene (Fig. 1) encodes only the neural cell adhesion molecule L 1 since the ATG of the GFAP gene was mutated and the L 1 coding sequence is followed 3' by a translational stop and a polyadenylation signal (Toggas et al. (1994) Nature 367:188-193). This construct was used to establish
  • mice Ll cDNA (Moos et al. (1988) Nature 334:701-703) was inserted into exon 1 of the murine glial fibrillary acidic protein (GFAP) gene modified as described previously (Toggas et al. (1994) Nature 367:188-193).
  • GFAP murine glial fibrillary acidic protein
  • the 4.05 kb mouse Ll cDNA containing the entire coding sequence of the protein and 250 3 ' 20 non-translated nucleotides was fused with the modified GFAP-L 1 transgene.
  • the 14.5 kb GFAP-L 1 transgene was excised from a modified cloning vector by digestion with Sfi I. followed by electrophoresis and electroelution from an agarose gel. Purified DNA was diluted to a final concentration of 2 ⁇ g/ml in T 5 E 0 , (5 mM Tris-HCI, pH 7.4. 0.1 mM EDTA). Approximately 2 pi of diluted DNA were 25 microinjected into the male pronucleus of fertilized eggs derived from CB6F1 females (superovulated) mated to C57B1/6J males. Eggs surviving the micromanipulation were transferred into oviducts of pseudo-pregnant foster mothers ,_. « , ⁇ M 96/32959
  • EXAMPLE 2 Southern blot analysis Mice were analyzed for the integration of the transgene into the mouse genome by Southern blot analysis of genomic DNA isolated from tail biopsies (Southern (1975) J Mol. Biol. 98:503-517). Transgenic founder mice were mated and pups screened in the same manner to establish transgenic lines. Ten ⁇ g samples of DNA were digested with either Bam HI or with Eco RI and Xba I followed by electrophoretic separation on a 0.7% agarose gel and transfer to Hybond N+ membrane (Amersham) under alkaline conditions.
  • a 3.3 kb Eco Rl-fragment of the Ll cDNA or a 330 bp Hind III fragment of SV40 late splice and polyadenylation site purified from A 1.5 plasmid (Maxwell et al. (1989) Biotechniques 7:276-280) were labelled with 32 ⁇ -CTP by random priming (Boehringer Mannheim) for use as probes. Prehybridization was performed at 65°C for one hour in 5x SSPE, 5x Denhardt's solution, 0.5% (w/v) SDS and 0.1 mg/ml sonicated non-homologous DNA. Hybridization was performed overnight. Final stringency wash conditions for all Southern blots were O. lx SSPE and 0.1% SDS (w/v) at 65°C.
  • RNA yields were estimated from absorbance at 260 nm. Ten ⁇ g of the RNA were 43 fractionated on 1 % agarose-formaldehyde gels for Northern blot analysis (Thomas (1980) Proc. Natl. Acad. Sci. USA 77:201-205).
  • Randomly primed L 1 cDNA probes were used to simultaneously detect the endogenous Ll mRNA of 6 kb (Tacke et al. (1987) Neurosci. Lett. 82:89-94) and the transgene-derived Ll mRNA of 4.2 kb. Densitometric analysis of Northern blots was performed on scanned images (Arcus scanner, Agfa-Gavaert) of the original films using the Image Program (NIH, Research Services Branch, NIMH).
  • L 1 transcripts of a size (4.2 kb) expected for transgene-derived mRNA (Fig. 2.). These transcripts are clearly distinct from the endogenous Ll mRNA which is 6 kb and derived from postmitotic neurons. Densitometric analysis revealed that the levels of transgene-derived Ll mRNA were 34%, 13% and 8% in lines 3426, 3427 and 3418, respectively, as compared to the levels of endogenous Ll mRNA (rated 100%).
  • control animals were taken from stocks of age-matched normal C57bl/6J mice or non-transgenic littermates.
  • small cerebellar neurons For isolation of small cerebellar neurons and for preparation of astrocyte cultures six-day-old ICR non-transgenic pups were used.
  • Dorsal root ganglion (DRG) neurons were prepared from eight- day-old chick embryos. 44
  • optic nerves were analyzed by in situ hybridization.
  • the optic nerve was chosen since it contains only glial cells and is free of neuronal cell bodies. Astrocytes in vivo normally lack expression of Ll at any developmental stage (unpublished data).
  • L 1 mRNA For detection of L 1 mRNA in cryostat sections of fresh-frozen brain sections, digoxigenin-labelled cRNA was generated by in vitro transcription (D ⁇ rries et al. (1993) Histochemistry 99:251-262). The sequence encoding the extracellular part of Ll (Moos et al. (1988) was subcloned into the pBluescript KS+ (Stratagene) vector. Anti-sense and sense cRNA probes were generated by transcribing the Ll insert after linearization of the resulting plasmid with Xho I or Xba I, using the T7 and T3 promoters, respectively.
  • GFAP cRNA probes For generation of GFAP cRNA probes, a 1.2 kb fragment of GFAP cDNA (Lewis et al. (1984) Proc. Natl. Acad. Sci. 81 :2743- 2745; kindly provided by Dr. N.J. Cowan) encoding the N-terminus of the protein was subcloned into the pBluescript KS+ vector. Anti-sense and sense cRNA probes were generated by transcribing the resulting plasmid, linearized with Eco RI and Xho I. from the T3 and T7 promoters, respectively. To improve tissue penetration, anti-sense and sense probes were sized under alkaline hydrolysis conditions to obtain an average fragment length of about 300 nucleotides. In situ hybridization on sections of optic nerves prepared from adult (12-weeks-old) animals was performed as described by elsewhere (D ⁇ rries et al. (1993); Bartsch et al., J. Neurosci
  • L 1 transcripts were detected only in nerve cells of the retina but not in optic nerve, neither before (Fig. 3A) nor after a lesion (Fig. 3B).
  • Ll mRNA was expressed by glial cells of the optic nerves from transgenic mice (Fig. 3C). Ll mRNA positive cells were detectable in both the distal myelinated and the proximal unmyelinated parts of the nerve. The intensity of the hybridization signal was higher in the unmyelinated proximal part, when compared to the myelinated distal part of the nerve.
  • polyclonal and monoclonal antibodies were visualized by horseradish peroxidase conjugated goat anti-mouse or rabbit antibodies (Dianova, Hamburg, Germany).
  • primary antibodies were detected using fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated goat anti-rabbit and goat anti-mouse antibodies (Dianova).
  • Digoxigenin-labelled cRNA probes for in situ hybridization were visualized by alkaline phosphatase- conjugated Fab fragments to digoxigenin (Boehringer Mannheim).
  • cerebellar neurons were maintained on cryostat sections of lesioned and contralateral unlesioned optic nerves (Fig. 7).
  • Optic nerves of 6 to 16- week-old mice were prepared as described by Bartsch et al. (1989) J. Comp. Neurol. 284:451-462. In brief, lesioned and unlesioned optic nerves were embedded and frozen in serum-free, hormonally defined medium (Fischer (1986b) Neurosci. Lett. 28:325-329) using liquid nitrogen. Tissue sections (14 ⁇ m thick) were cut longitudinally on a Frigocut 270-cryostat (Jung-Reichardt), mounted onto poly-L-lysine-coated (Sigma, 0.001% in water) sterile glass coverslips and air-dried for 2-3 hours in a sterile chamber. After washing the sections for 5 minutes with medium.
  • Percoll gradient-purified small cerebellar 47 neurons (Keilhauer et al. (1985) Nature 316:728-730) from six-day-old ICR mice (6 x 10 4 cells in 100 ⁇ l medium) were applied to each coverslip. Cells were maintained in an incubator at 37°C with a humidified atmosphere of 5% CO, and 95% air.
  • Neurite outgrowth was also measured in the presence of antibodies. Sections were pre-incubated with polyclonal L 1 antibodies or polyclonal antibodies against mouse liver membranes (100 ⁇ g/ml, dialyzed extensively against and diluted in culture medium) for 1 hour at 37°C. After removal of antibodies, sections were washed carefully with culture medium (5 times, each for 5 minutes at room temperature) and Percoll gradient purified small cerebellar neurons were added. After 2 days, cryostat cultures were fixed in 4% paraformaldehyde in PBS for 30 minutes at room temperature and the neurite lengths were measured. To avoid "edge effects" in the measurements, we did not evaluate the sections which were situated in the outer rim comprising 20% of the coverslips.
  • Fig. 8 For transgenic animals, an increase in neurite length was observed on lesioned compared with unlesioned nerves. In contrast, neurite lengths on lesioned and unlesioned optic nerves of wild type animals were not significantly different (Fig. 8). Neurites of neurons cultured on unlesioned optic nerves from transgenic animals were consistently longer than neurites of neurons cultured on unlesioned nerves from wild type animals. A maximal increase in neurite length of about 300% was observed when using sections from line 3426. Similarly, neurite length on lesioned nerves of transgenic lines was increased up to 400% when compared with lesioned nerves from wild type animals.
  • the neurite outgrowth promoting activity of transgenic optic nerves correlated positively with the level of Ll expression (Fig. 8).
  • Unlesioned optic nerves of line 3426, which express the highest levels of Ll protein were more potent in increasing neurite outgrowth than those of lines 3427 and 3418 expressing, by comparison, lower levels of Ll (in decreasing order).
  • neurite outgrowth was four times higher than on lesioned optic nerves of wild type animals.
  • Contaminating oligodendrocytes and neurons were removed by shaking the flasks at every medium change and by subculturing the cells at intervals of four days. Immunostaining for GFAP after 14 days of maintenance showed that more than 90% of the cells were astrocytes. After 14 days in culture, the cells were trypsinized and maintained as monolayers for five days on poly-L-lysine-coated glass coverslips. Percoll gradient purified small cerebellar neurons (Schnitzer et al. (1981)) from six-day-old mice and dorsal root ganglion (DRG) (Seilheimer et al. (1988) J. Cell. Biol.
  • DRG neurons was also studied in monolayer cultures of astrocytes derived from transgenic (line 3426) or non-transgenic controls (Fig. 10, Table 1).
  • Neurite length of cerebellar or DRG neurons on transgenic astrocytes was approximately 15% or 50% higher, respectively, when compared with neurite length using wild type astrocytes (Table 1).
  • Anti-liver membrane antibodies did not affect neurite length on astrocyte monolayers from wild type or transgenic animals (Fig. 10, Table 1).
  • Pre-incubation of astrocyte monolayers with Ll antibodies did not significantly affect neurite length on cells from wild type animals.
  • Ll and GFAP immunostaining of fresh-frozen cross- or longitudinally sectioned optic nerves or astrocytic monolayers of wild type and transgenic animals were performed as described (Bartsch et al. (1989)).
  • double-labelling we first incubated astrocytes as live cells with Ll antibodies (2 ⁇ g/ml in 1% BSA in PBS) at 4°C for 30 minutes. After permeabilizing the cells with 70% methanol at -20°C for 10 minutes, cells were incubated with GFAP antibody for 30 minutes at 4°C.
  • the Aurion immuno R-Gent silver enhanced staining was used according to the manufacturer's instructions (Aurion, Immuno Gold Reagents & Accessories Custom Labelling, Wageningen, The Netherlands) with minor modifications.
  • cultures were fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature, incubated in 50 mM glycine in PBS for 10 minutes and then treated for 15 minutes in blocking buffer (BB, 0.5% BSA in PBS). After 3 washes in BB, each for 5 minutes, cells were incubated with Ll antibodies diluted in BB (2 ⁇ g/ml) for 30 minutes at room temperature.
  • cultures were washed 3 times in BB each for 5 minutes and secondary antibody diluted 1:20 in BB was added for 1 hour at room temperature. After 3 washes with distilled water, cultures were fixed in 2% glutaraldehyde in PBS for 10 minutes at room temperature and washed 3 times with distilled water. A 1 : 1 mixture of enhancer and developer was then added at room temperature. After the appearance of the reaction product, coverslips were washed 3 times with distilled water and embedded in glycerol.
  • Ll expression was additionally analyzed in cultures of astrocytes prepared from forebrain of six-day-old transgenic animals. No L 1 immunoreactivity was detectable on astrocytes from wild type animals (Fig. 5D). In contrast, Ll positive cells were present in cultures from transgenic animals (Fig. 5A). As demonstrated by double-immunostaining, the same cells also proved positive for GFAP (Fig. 5B and E) indicating that the cells expressing Ll are indeed astrocytes. Since Ll immunostaining was performed on living cells, it seems likely that in the transgenic animals L 1 is also exposed on the cell surface of astrocytes in vivo.
  • Horseradish peroxidase-conjugated secondary antibody (2 ⁇ g/ml) was detected by the ECL Western blotting detection kit (Amersham). Densitometric analysis of immunoblots was performed on scanned images (Arcus scanner, Agfa- Gavaert) of the original films using the Image Program (NIH, Research Services Branch, NIMH).
  • L 1 expression in unlesioned optic nerves of transgenic animals was about 40% and 13% (lines 3426 and 3427, respectively) higher than in unlesioned optic nerves of wild type animals.
  • Ll expression in lesioned transgenic nerves was 310% and 200% (lines 3426 and 3427, respectively) higher as compared with lesioned nerves of wild type animals.
  • a comparison between lesioned and contralateral unlesioned optic nerves from wild type animals revealed a decrease in L 1 protein expression of about 40% on the lesioned side.
  • the amount of L l protein in lesioned nerves of lines 3427 and 3426 increased by approximately 30% when compared with the unlesioned contralateral side.
  • the expression level of Ll in line 3426 was approximately 35% and 25% higher than in the line 3427 for unlesioned and lesioned optic nerves, respectively.
  • L2/HNK-1 immunoreactivity in reinnervated peripheral nerve preferential expression of previously motor axon-associated Schwann cells
  • L2 The carbohydrate epitope L2/HNK-1 (hereafter designated L2) is expressed in the adult mouse by myelinating Schwann cells of ventral roots and muscle nerves, but rarely by those of dorsal roots or cutaneous nerves. Since substrate-coated L2 glycolipids promote outgrowth of cultured motor but not sensory neurons, L2 may thus influence the preferential reinnervation of muscle nerves by regenerating motor axons in vivo.
  • Myelinating Schwann cells previously associated with motor axons thus differed from previously sensory axon-associated myelinating Schwann cells in their ability to express L2 when contacted by motor axons.
  • This upregulation of L2 expression during critical stages of reinnervation may provide motor axons regenerating into the appropriate, muscle pathways with an advantage over those regenerating into the inappropriate, sensory pathways.
  • L 1 Given the role of L 1 in mediating cell-cell contact, the present study was undertaken in order to determine if L 1 is amongst the learning-associated glycoproteins participating in either or both waves of glycoprotein synthesis, and is necessary for memory formation. If so. antibodies to Ll administered at an appropriate time relative to training should prevent the synaptic remodelling necessary for long term memory and therefore produce amnesia for the task. Similarly, if the extracellular domains of the L 1 molecule play a part in the recognition and adhesion processes which are required for synaptic remodelling and stabilization, exogenously applied extracellular domain fragments which will bind homophilically to the endogenous molecule might disrupt this process.
  • Antibodies and Fragments Polyclonal antibodies were prepared in rabbits by immunization with immuno- affinity purified Ll (Ng-CAM, 8D9) following an established immunization procedure (Rathjen et al. (1984)). Ll was isolated from one-day old chicken brains using an 8D9 monoclonal antibody (Lagenaur and Lemmon (1987) Proc. Nat 'I Acad. Sci. USA 84:77533-7757) column again using established procedures (Rathjen et al. (1984)). Antibodies were isolated from the serum obtained after the third immunization using Protein G Sepharose (Pharmacia LKB) according to the manufacturer's instructions. Recombinantly expressed fusion proteins in E. coli representing the six immunoglobulin-like (Ig-I-VI) and five fibronectin type III homologous repeats (FN1-5) were prepared as described by Appel et al (1993).
  • FN1 -5 and Ig I-VI fragments were dialyzed overnight against 0.9% saline and the concentration adjusted to 1 mg/ml for L l and 250 ⁇ g/ml for the fragments.
  • Chicks received bilateral intracranial injections into the intermediate medial hyperstriatum ventrale (IMHV) of 10 ⁇ l Ll antibodies per hemisphere; control animals received similar injections of saline.
  • IMHV intermediate medial hyperstriatum ventrale
  • Accurate delivery into the IMHV was received by the use of a specially designed head holder and sleeved Hamilton syringe (Davis et al. (1982) Pharm. Biochem. Behav. 17:893-896).
  • a previous report has demonstrated (Scholey et al. (1993)) that there is a slow diffusion of antibody from the injection site in the hours following injection. The accuracy of placement of the injection was routinely monitored by , negligence,_ « . « « 6/32959
  • Ll fragments FNl-5 and Ig I-IV were injected at either -30 minutes or +5.5 hours and retention tested at 24 hours. Retention in groups of saline and Ll -antibody or L 1 -fragment-injected chicks was compared statistically by ⁇ 2 . Results are shown in Figures 13 and 14.
  • Transverse hippocampal slices 400 ⁇ m) from halothane-anaesthetized male Wistar rats (180-220g) were prepared using standard techniques. Slices were maintained in an interface chamber and initially allowed to recover for 45 min. in a hyperosmolar (320 mOsm/kg) artificial cerebrospinal fluid (ACSF) at room temperature.
  • a hyperosmolar 320 mOsm/kg
  • an artificial cerebrospinal fluid ACSF
  • the bath temperature was then raised to 30°C and the medium was changed to a normotonic ACSF (307 mOsm/kg) containing (in mM): NaCl, 124.0; KCl, 2.5; MgSO 24 , 2.0: CaCl,, 2.5; KH,PO 4 , 1.25; NaHCO 3 , 26.0; glucose, 10; sucrose, 4; bubbled with 95% O,/5% CO, (pH 7.4); perfusion rate: 0.75 ml/min.
  • the Schaffer collateral/commissural fibers were stimulated by twisted platinum- iridium wires (50 ⁇ m diameter) placed in the stratum radiatum of the CAl region.
  • Test stimuli consisted in monophasic impulses of 100 ⁇ s duration every 30 seconds and the stimulus strength was adjusted to obtain 30% of the maximal EPSP amplitude (maximal EPSP without superimposed population spike).
  • EPSP ' s were recorded from the CAl stratum radiatum by means of 2 glass micropipettes (2 M 59
  • NaCl, 1-5 M ⁇ positioned about 300 ⁇ M apart from the stimulation electrode on each side.
  • LTP was induced with a theta burst stimulation (TBS) paradigm consisting of three trains spaced by 4 seconds; each train consisted of ten high frequency bursts of 5 pulses at 100 Hz and the bursts were separated by 200 ms (Reichardt et al. (1991) Annu. Rev Neurosci. 14:531-570). Duration of the stimulation pulses was doubled during TBS.
  • TBS theta burst stimulation
  • D-AP5 D(-)-2-amino- 5-phosphonopentanoic acid
  • D-AP5 D(-)-2-amino- 5-phosphonopentanoic acid
  • Whole cell recordings were obtained from CAl neurons using the "blind" patch clamp method with an EPC-9 patch clamp amplifier. The bath temperature was 30°C. Patch electrodes were pulled from 1.5 mm OD borosilicate glass and had resistances between 3 and 8 M ⁇ . The pipettes were neither fire polished nor coated. The electrodes were routinely filed with a solution containing (in mM): potassium gluconate. 129; KCl.
  • Ig-like domains I-VI and FN type III homologous repeats I-V of L 1 were expressed in bacteria and purified as described (Hynes et al. (1992)). Antibodies to NCAM and axonin-1 were produced as described (Larson et al. (1986) Science 232:985- 988; Bailey et al. (1992) Science 256:645-649). Production of oligomannosidic glycopeptides from ribonuclease B and control glycopeptides from asialofetuin have been described (Larson et al. (1986)). Results are shown in Figure 16.
  • NMDA receptor-mediated EPSP's were isolated by applying 30 ⁇ M of the non- NMDA blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Tocris) starting 20 minutes prior to the application of antibodies or glycopeptides. At the end of each experiment, it was verified that D(-)-2-amino-5-phosphonopentanoic acid (D-AP5; 30 ⁇ M; Tocris) completely suppressed these responses. Results are shown in Figure 17.
  • Soluble Ll (Ll-Fc) is functionally active and is a potent agent in neuronal survival
  • Soluble Ll was made in COS cells as a recombinant Ll-Fc fusion protein by the procedure described in Neuron 14:57-66, 1995.
  • the recombinant protein was purified by Protein A affinity chromatography, and was used either as a substrate coated onto plastic or as a soluble molecule added to the culture medium at approximately 1-10 ⁇ g/ml.
  • Neurite outgrowth and survival of mesencephalic neurons from day 17 rat embryos were examined in culture after 7 days in vitro maintenance.
  • Dopaminergic neurons were recognized by immunostaining for dopamine- ⁇ -hydroxylase (DBH) and quantified using IBAS morphometric equipment.
  • DBH dopamine- ⁇ -hydroxylase
  • Recognition among neural cells is an important prerequisite for the development of a functioning nervous system.
  • Recognition molecules are expressed at the cell surface, where they mediate interaction between neighboring cells, like cadherins. or between the cell surface and the extracellular matrix, like integrins (Takeichi. 1991; Ruoslahti. 1988: Hynes. 1992).
  • the most prominent family of recognition molecules comprises immunoglobulin (Ig)-like domains.
  • the Ig-like domains reflect a common ancestry of immunoglobins and cell adhesion molecules, both of which are involved in specific recognition events (Edelman. 1970).
  • the Ig superfamily comprises by now more than two dozen distinct molecules.
  • Ig-like domain containing molecules have multiple functions within the extracellular domain: Receptors for cytokines and neurotrophins have high affinity receptive functions as well as recognition properties (Tannahill et al.. 1995; Pulido et al.. 1992).
  • the three-dimensional structure of Ig-like domains is similar to FN-like xrp_vus (Mam £t al.. 1992; Leahy et al.. 1992). which are also structural motifs in several extracellular matrix molecules, such as fibronectin. members of the tenascin family, and others CWilliams and . 1988: Baron et al.. 1992: Erickson. 1993).
  • Neural recognition molecules of the Ig supefamily have characteristic temporal, spatlel. and cell-type specific espression patterns (for reviews, see Edelman. 1988: Schachner. 1991. 1994: Rathjen and Josseii. 1991 : 63
  • Recognition molecules of this family are functionally overlapping in that all promote cell adhesion and neurite outgrowth. Some recognition molecules are strongly homophilic, i.e. self binding partners, whereas others are predominantly heterophilic, i.e. they bind to non-self partners which often comprise other members of the Ig superfamily or extracellular matrix molecules (Brummendorf and Rathjen, 1993, 1994).
  • neural recognition molecules of the Ig superfamily the family of molecules related to the neural recognition molecule L 1 shows striking similarity in function and structure. They are potent neurite outgrowth promoters and are expressed relatively late during development, mostly at the state when axogenesis occurs. They are predominantly expressed by neurons, although some members of the L 1 family are also present on neurite outgrowth promoting glial cells (Martini and Schachner. 1986: Bixby et al.. 1988; Seilheimer and Schachner, 1988).
  • CHLl close homolog of Ll
  • CHLl Ll
  • FN-like repeats of which four are highly homologous to the FN- like repeats of other Ll family members.
  • the partial FN-like repeat localizes to the membrane-adjacent region of the molecule, which is the most variable region among L 1 related molecules.
  • Other features of CHL 1 shared with members of the Ll family are its predominant and developmental ly late expression in the nervous system, and its high level of N-glycosylation, including expression of the HNK- 1 carbohvdrate. 6
  • ICR mice and Wistar rats were used for tissue preparations.
  • N-CAM. and MAG were immunoaffinity purified from detergent extracts of crude membrane fractions from adult mouse brain using monoclonal antibody columns (Rathjen and Schachner, 1984; Falssner et al., 1985; Poltorak et al., 1987).
  • RNA Eight micrograms of poly(A) * RNA were used for synthesis of oligo(dT)-primed double- stranded cDNA using a cDNA synthesis kit (Amersham).
  • the cDNA was size- selected and ligated into the plasmid pXMDl with Dralll-adaptors containing a Sail site (Kluxen et al.. 1992).
  • E. coli strain TOP 10 Invitrogen, Netherlands was used.
  • For screening aliquots were directly plated onto Nylon membranes (BIODYN ⁇ TM Pall) with a density of about 2xl0 4 bacteria/filter (138 cm 2 ). Replica filters were incubated overnight at 37°C.
  • the bacteria were lysed (0.5M NaOH; 1.5M NaCl), filters were neutralized (3M NaCl; 0.5M Tris-HCI pH 8.0), washed in a 2xSSC, air-dried, and baked for 2 hr. at 80°C.
  • the Nylon membranes were prehybridized, hybridized with a 1 kb fragment (HincII/Kpnl) of CHLl (derived from the ⁇ gtl l library) redioiabelled by random-priming (Boehringer Mannheim) according to the manufacturer ' s protocol, washed under high-stringency conditions at 42°C.
  • the 4.43 kb insert of clone pX#2 was ligated into Sail digested pXMDl (Kluxen et al., 1992. Kluxen and Lobbert, 1993).
  • a subfragment (EcoRI (plasmid polylinker)/Pvull bp 4048) of the mouse Ll cDNA (Moos et al.. 1988) was treated with T4 DNA polymerase and ligated into pXMDl .
  • COS-1 cells were maintained in DMEM (0.1% glucose) supplemented with 10% (v/v) fetal calf serum at 37°C in a humidified atmosphere with 5% CO,.
  • DEAE dextran-mediated DNA transfection was performed as described (Kluxen et al., 1992) with some modifications. Briefly, the cells were seeded at about 10,000 cells/cm 2 .
  • DMEM fetal calf serum
  • DMEM fetal calf serum
  • transfection solution composed of DMEM supplemented with 10% (v/v) Nu-serum (Becton Dickinson, Switzerland), 0.4 mg/ml (w/v) DEAE-dextran (Pharmacia). 50 ⁇ M chloroquine.
  • PC 12 cells were maintained in DMEM with 10% (v/v) fetal calf serum and 5% (v/v) horse serum on collagen coated tissue culture dishes.
  • NGF nerve growth factor
  • the medium was removed from monolayers at about 50% confluency and replaced with medium of reduced serum content (5% horse serum) supplemented with 100 ng/ml 7s-NGF (Sigma, Switzerland). After two days of incubation the cells were detached by incubation with 0.1% trypsin and 0.04% EDTA, collected and subjected to RNA extraction.
  • astrocytes Primary cultures of astrocytes were prepared according to McCarthy and De Vellis (1980) with modifications (Guenard et al. 1994) and used for immunostaining after one to two weeks in vitro. Primary cultures of oligodendrocytes were prepared as described by Laeng et al. (1994) and maintained in vitro for 12 days.
  • the 4.43 kb insert of clone pX#2 was ligated into Sail digested pBS II SK. (Stratagene) followed by deletion of an Apal (vector)/ A vrll (bp 3330 ( Figure 18)) fragment to obtain the cDNA fragment of CHLl encoding the extracellular part of the protein (see Figure 18 and 19).
  • a similar construct for L 1 was prepared by Iigation of an EcoRI (plasmid polylinker)/EcoNI (bp 3304) fragment of the L l cDNA (Moos et al.. 1988) treated with T4 DNA ploymerase and ligated into Smal digested pBS II SK-.
  • the plasmids were digested with Xbal and used for synthesis of j2 P-labeIed antisense RNA with T7 RNA polymerase as described (Melton et al., 1984).
  • mRNA was prepared from different tissues of neonatal and 9-day-old mice using the OligotexTM Direct mRNA-Method (QIAGEN Inc., D ⁇ sseldorf, Germany) following the manufacturer ' s instructions.
  • RNA transferred and bound to the membrane was controlled by methylene blue staining (Sambrook et al., 1989). Following prehybridization for 2 hr at 65°C, the membrane was hybridized overnight using CHL 1 - and L 1 - specific 32 P-labeled antisense RNA probes in hybridization buffer (5xSSC, 2.5xDenhart's solution, 50 mM Na,PO, (pH 6.5). 0.1% SDS, ImM EDTA. 2 ⁇ g/ml salmon sperm DNA, 50% formamide) at 65°C. The filter was then washed three times at 65°C in O. lxSSC, 0.1% SDS for 1 hr and exposed to X-ray film.
  • hybridization buffer 5xSSC, 2.5xDenhart's solution, 50 mM Na,PO, (pH 6.5). 0.1% SDS, ImM EDTA. 2 ⁇ g/ml salmon sperm DNA, 50% formamide
  • a 1.7-kb cDNA-fragment of CHLl (Mscl; bp 1791 (which originates from the vector cloning site and the 5' end of the ⁇ gtl 1 derived CHLl clone) and BsmAl; bp 3494) encoding the 6th Ig-like domain (IgVl ) and FN-like repeats 1.4.5 (see Figures 18 and 19b) was subcloned into the unique BamHI restriction site of the pET- vector (Studier and Moffatt, 1986). The correct sequence of the plasmid was confirmed by sequencing. E. coli strain BL21 (DE3) was transformed with this plasmid.
  • Detergent lysales of whole tissue were prepared by homogenization of tissues in 40 mM Tris-HCI (pH 7.4), 150 mM NaCl, 5 mM EDTA. 5mM EGTA, ImM phenylmethysulfonylfluoride (PMSF), 1% Triton X-100 and maintained at 4°C for 3 hr under constant stirring. The soluble fraction was separated from insoluble material by centrifugation at 100,000 g.
  • Tris-HCI pH 7.4
  • PMSF ImM phenylmethysulfonylfluoride
  • tissues were homogenized in 1 mM NaHCO 2 (pH 7.9), 0.2 mM CaCl 2 , 0.2 mM Mgcl,, 1 mM spermidine, 5 ⁇ g/ml aprotinin. 10 ⁇ g/ml soybean trypsin inhibitor. 1 mM PMSF, and 0.5 iodoacetamide at 4°C.
  • Membrane and soluble fractions were then separated and the membrane pellot was resuspended in solubilization buffer (20 mM Tris-HCI (pH 7.9). 0.15 M NaCl.
  • Transiently transfected COS-1 cells were washed twice with HBSS and incubated with 1 mM EDTA in HBSS for 10 min at 37°C. The cells were then detached with a fire polished Pasteur pipette and collected by centrifugation at 200g for 10 min at 4°C. The cells were lysed in 20 mM Tris-HCI (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM iodoacetamide, 1 mM PMSF, and 1% MP-40 and the supernatant was cleared by centrifugation (13000g). Protein determinations were performed as described by Bradford (1976).
  • Bound antibodies were either detected by the enhanced chemiluminescence (ECL) method according to the manufacturer ' s instructions using ECL Western blotting detection reagents (Amersham) and X-ray films, or by using BCIP and NBT as chromogenic substrates.
  • ECL enhanced chemiluminescence
  • EXAMPLE 22 Enzyme-linked immunosorbent assay The enzyme-linked immunosorbent assay (ELISA) was performed as described by Husmann et al. (1992) with the exception that proteins were coated at concentrations of 100 ng/ml. CHLl antiserum was used in several dilutions between 1 :250 to 1:2X10 6 .
  • CHLl Detergent lysates of brain tissue homogenate (200 ⁇ l. 6 mg/ml protein concentration) from seven-day-old mice were separated into a soluble fraction and insoluble material by centrifugation (see Tissue fractions) and incubated with 0.5 units N-glycosidase F or 2.5 units O-glycosidase, or both enzymes at these concentrations according to the manufacturer ' s instructions (Boehringer Mannheim, Germany). The lysates were resolved by SDS-PAGE on 10% gels. The proteins were transferred to nitrocellulose and incubated with CHLl antiserum (1:500 diluted) directed against the recombinant CHLl protein fragment (see Figure 18).
  • the Sepharose beads were boiled for 10 min in 5x sample buffer (250 mM Tris-HCI (pH 6.6), 10% SDS, 50% glycerol, 0.5% bromophenol blue, 25% B-mercaptoethanol) and the supernatant was resolved by SDS-PAGE on 5 10% gels.
  • sample buffer 250 mM Tris-HCI (pH 6.6), 10% SDS, 50% glycerol, 0.5% bromophenol blue, 25% B-mercaptoethanol
  • the proteins were transferred to nitrocellulose and detected with polyclonal antibodies against Ll. CHLl antiserum, or monoclonal antibody 412 by Western blot analysis.
  • CHL1-, and mock (vector 10 only)- transfected COS-1 cells plated on coverslips were incubated for 30 min at room temperature with primary antibody (CHLl antiserum (1 :100 diluted) or Ll polyclonal antibodies (1:200 diluted)) in DMEM containing 10% fetal calf serum, 10 mM Hepes (pH 7.3), and 0.02% NaN 3 , and then with secondary antibody. After innunostaining, the cells were fixed with 4% paraformaldehyde in phosphate 15 buffered saline (pH 7.3) and mounted in Moviol (Hoechst) containing 2.5% potassium iodide.
  • clone 31 1 Screening of a ⁇ gtl 1 expression library for cDNA clones encoding the cell adhesion molecule L 1 with polyclonal antibodies raised against brain-derived immunopurified Ll (Tacke et al. 1987) identified the clone 31 1. It contained a partial cDNA homologous to Ll (34.1% according to Lipman and Pearson (1985)) and an open reading frame of 21 12 base pairs (bp) coding for 704 amino acids including the cytoplasmic part. To isolate full length cDNA clones, a DNA fragment of this clone was used for screening a different cDNA library. Six independent clones were isolated. Two clones contained 4.2 and 4.4 kb inserts comprising the entire coding region of a close homolog of Ll (CHLl). The clone containing the 4.4 kb insert was further investigated.
  • the 4.4 kb insert encodes a 5' untranslated region of 295 bp. an open reading frame of 3627 bp. and a 3' untranslated region of 518 bp (Figure 18). Although there is an oligo(A) tract at its 3 ' terminus, a clear consensus polyadenylation signal upstream of this sequence is missing.
  • the flanking sequences of the AUG start codon do not conform to the optimal consensus sequence for initiation of translation (Kozak, 1987). However, this AUG is taken as the start codon for translation based on two lines of evidence.
  • the putative extracellular domain is composed of 1081 amino acids, with 18 potential sites for N-glycosylation ( Figures 18 and 19a) and more than 60 potential O-glycosylation consensus sites (not shown) (Pisano et al., 1993). followed by a transmembrane domain of 23 amino acids, as judged by hydropathy analysis according to Kyte and Doolittie (1982) ( Figure 19c). This domain is flanked at its N-terminal end by a polar residue and at its C-terminal end by a basic amino acid, consistent with a stop transfer signal ( Figure 18). The intracellular region is composed of 105 amino acid residues.
  • the extracellular region contains the two major structural motifs of repeated domains that are characteristic of the Ll family: a 685 amino acid stretch with homology to Ig-like domains and a 472 amino acid stretch with homology to FN- like repeats ( Figures 1 and 2a). All of the six Ig-like domains contain the characteristic pair of cysteine residues located at 47-54 amino acids apart from each other ( Figure 19). A conserved proline (except in the sixth Ig-like domain) at the end of ⁇ -strand B in conjunction with a C2-type cluster of conserved amino acids around the second cysteine residue in each domain (DXGXYXCXAXN) assign the Ig-like domains to the C2-set (Williams and Barclay, 1988).
  • the Ig-like domains and the membrane spanning region are four domains that are homologous to the FN-like repeats in fibronectin (Kornblihtt et al., 1985). Each of these domains of approximately 100 amino acids contains the highly conserved tryptophan (except for the first FN-like repeat) and tyrosine/phenylalanine residues in the N- and C-terminal regions, respectively.
  • the fifth FN-like repeat is, in contrast to the other members of the L 1 family, only a rudimentary one-half FN-like repeat (Figure 18).
  • this half FN-like repeat represents one of several alternatively spliced forms, one of which contains a full FN-like repeat, remains to be determined by other methods than Northern blot analysis. It is noteworthy in this context that no evidence for alternative splicing was found by restriction analysis of the six independently isolated clones (not shown). Alternative splicing of the fifth FN-like domain was observed for Nr- CAM/BRAVO, where cDNA isoforms were isolated lacking the fifth FN-like repeat (Grumet et al.. 1991 : Kayyem et al.. 1992.) The absence of the fifth FN- like domain in chicken neurofascin (Volkmer et al..
  • CHL 1 Another structural feature of CHL 1 is the presence of an RGD sequence (amino acids 185-187) in the second Ig-like domain ( Figure 18.
  • RGD sequence amino acids 185-187
  • This tripeptide has originally been identified as a cell attachment site within the tenth type III domain of fibronectin (Pierschbacher and Rusolahti, 1984) and contributes to integrin binding (for review see Rusolahti and Pierschbacher, 1987).
  • Three dimensional structure analysis of FN-like repeats showed that the RGD motif is localized between the ⁇ -strands F and G (Main et al., 1992). This motif is also found in other members of the L 1 family.
  • the RGD sequence is found at the same position, between the ⁇ -strands F and G.
  • RGD motifs are also found in Ll (two in Ll mouse and rat (NILE), and one in human L l (Moos et al., 1988; Hlavin and Lemmon, 1991 ; Prince et al., 1991). All Ll RGD sequences are found in the sixth Ig-like domain, but in a different amino acid environment than RGDs in the FN-like modules of fibronectin.
  • the tripeptide in CHL 1 is localized on the ⁇ -strand E of the second Ig- like domain. Whether the RGD sequences in these proteins are functionally active is currently not known. It is noteworthy in this context that neurite extension induced by TAG-1 (Furley et al., 1990), a member of the F3/F11 family (Br ⁇ mmendorf and Rathjen, 1993, 1994) that contains a RGD motif in the second FN-like domain depends on B, integrin and Ll (Felsenfeld et al.. 1994). This observation raises the possibility of a direct physical interaction between the second FN-like repeat of TAG-1 and ⁇ , integrin.
  • CHLl also contains a DGEA sequence (amino acids 555-558) in the ⁇ -strand C of the sixth Ig-like domain ( Figure 18). This sequence is not found in other members 75 of the Ll family.
  • the DGEA sequence has also been implicated in ⁇ , ⁇ , integrin recognition of type I collagen containing this motif (Staatz et al., 1991).
  • mice, human, and rat Ll/NILE The sequences of mouse, human, and rat Ll/NILE, chicken Ng-CAM, chicken Nr- CAM, zebrafish Ll.l (Tongiorgi et al., 1995), chicken neurofascin/rat ABGP, Drosophila neuroglian (Bieber et al., 1989), mouse F3/chicken Fl /human CNTN1 (Gennarini et al..
  • TABU 2 C ⁇ npu ⁇ oo of t tftt-rt timibritiet af ⁇ txTtocrBnt-r puti of Ll relaie ⁇ wteculri
  • the intracellular regions of the members of the Ll family were compared with each other and with other neural cell adhesion molecules of the Ig superfamily. Values indicate the percentage of ami acid identity after alignment according to Hein (1990). The species are indicated in brackets! c - chicken, d - Orosphila, h - human, m * mouse, r » rat, and zf ⁇ zebrafish. Mouse, rat, and human Ll are identical. Mouse NrC and neurofascin are partial sequences of 91 and 86 amino acid residues, respectively.
  • T e tab e shows t e num er o res ues etween conserve am no ac s w t n t e g- e oma ns (cyste ne ⁇ involve in the S-S bridges: Igl, Ig2, Ig3, Ig4, Ig5, and Ig6), FN-like repeats (tryptophan of the second 0-strand and tyro ⁇ ine/phenylalanine of the sixth ⁇ -strand: FN1, FN2, FN3, and FN6), and between these domains (1-2, 2-3, 3-4, 4 5, and 5-6; FN1-FN2, FN2-FN3, and FN3-FN4).
  • the distance between the conserved amino acids of the second cysteine of the sixth Ig-like domain and the first tryptophane of the first FN-like repeat (Ig6-FN1) reflects the distance between the Ig-like domains and FN-like repeats.
  • Th average and standard deviation (SD) of the individual distanc for the Ll related molecules are indicated, DCC, HLAR, rse, NCAM, MAG, neuro usculin, and fibronectin (III) are given as a control.
  • Siilarity index is given at the right margin. 7 ⁇ no conserved amino acid, * ⁇ not used for average and SD calculations.
  • CHLl is most similar to chicken Ng-CAM (37% amino acid identity in the extracellular domain. Table 1 ) and mouse Nr-CAM (64% amino acid identity in the intracellular domain. Table 2). However, the degree of identity, particularly in the extracellular part, is not sufficient to consider these proteins as species homologs. Recently, a partial cDNA clone of mouse Nr-CAM (Moscoso and Sanes, 1995) was identified. Mouse Nr-CAM is nearly identical (99%) to chicken NR-CAM (see Table 2). Therefore. CHLl is not likely the Nr-CAM homolog in the mouse.
  • CHL 1 is the fourth member of the L 1 family in the mouse with L 1 , Nr-CAM, neurofascin (Moscoso and Sanes, 1995), and CHLl , with a highly conserved species homolog in human (Adams et al., 1992, 1993).
  • mice L 1 and chicken Ng-CAM are species homologs, since they show only 01% sequence identity in the intracellular domain (Table 2). Rather, the existence of Ng-CAM as the fifth member of the Ll family in the mouse with a highly conserved intracellular domain is to be expected.
  • mouse L 1 upon heterophillc interaction with chicken Ng-CAM promotes neurite outgrowth (Lemmon et al.. 1989). suggesting that members of the Ll family may interact with each other.
  • Molecules containing six Ig-like domains and at least four FN-like repeats reveal a very constant number of amino acids separating these conserved amino acids. Five different distance parameters were considered:
  • FxVxAxNxxG(8x)S(4x)TxxAxPxxxP at the end of the first FN-like repeat or NxxGxGPxS between the last two ⁇ -strands of the third FN-like repeat support the notion that F3 belongs to the L 1 family.
  • the number of amino acids between adjacent domains is even more conserved among these molecules, indicating that the distance between the individual domains is an important structural feature, i.e. critical for functioning of neural recognition molecules (Table 3. columns 1-2, 2-3, 3-4, 4-5. 5-6, FN1-FN2. FN2-FN3, and FN3-FN4).
  • this high conservation of the order (colinearity) and spacing may be used to define more generally the extracellular domain of the Ll family members. With the criteria just defined, these contain a module of six Ig-like domains at the N-terminus followed by four FN-like repeats. We would like to call this structural feature the Ll family cassette.
  • L 1 family shares the characteristic features of the L 1 family cassette and. additionally, highly conserved amino acids.
  • the general Ll family may thus be subdivided into the "classical" Ll family members (Ll. CHLl, Ng-CAM, Nr-CAM. neurofascin, neuroglian, Ll .
  • N-CAM Non-CAM-binding protein
  • MAG Arquint et al., 1987; Lai et al., 1987; Salzor et al., 1987
  • neuromusculin Kanla et al., 1993
  • rse Mark et al., 1994
  • fibronectin Kernblihtt et al., 1985
  • Ig-like domains and/or FN-like repeats show clearly distinct distance parameters, indicating that they are much less related to each other and to the members of the Ll family (Table 3).
  • HLAR human leukocyte common antigen-related gene
  • DCC tumor suppressor gene product
  • RNA The 8 kb RNA was detected in cerebellum, brain minus cerebella, and spinal cord but not in dorsal root ganglia (DRG) ⁇ Figure 21a).
  • DRG dorsal root ganglia
  • the Ll riboprobe showed a strong signal with RNA from DRG ( Figure 21a).
  • CHLl mRNA was also detectable in nine-day-old rat cerebellum and six- day-old rat spinal cord but not in rat PC 12 cells maintained with and without NFG or in COS-1 cells ( Figure 21b). In all other tissues analyzed (thymus, lung, liver, intestine, spleen, and kidney) no signal was detectable (Figure 21a).
  • the CHL 1 antisera were used to identify immunoreactive proteins in several tissues (brain, liver, lung, kidney, and intestine from nine-day old mice. Figure 23b). Crude membrane fractions, soluble and insoluble in 0.5 % Triton X-100, were analyzed by Western blotting. Polyclonal antibodies against Ll were used as a control. The CHLl antibodies recognized three distinct bands of 185, 165, and 125 kD in the insoluble and soluble fractions of brain membranes.
  • the 185 kD band was only weakly detectable in the soluble fraction and the 125 kD band was less prominent in the insoluble fraction ( Figure 23b, lane 1 and 2), indicating that the 185 kD band is probably the membrane bound form of CHLl, whereas the 125 and 165 kD forms are probably proteolytically cleaved fragments.
  • a similar pattern of immunoreactive bands was observed after Western blot analysis of CHL 1 transfected COS cells and total brain tissue (not shown). A similar pattern of bands has been observed for Ll (Faissner et al., 1985: Sadoul et al.. 1988), Ng ⁇ CAM (Grumet et al..
  • Glial cells in the optic nerve did not contain detectable levels of Ll transcripts (Fig. 24a).
  • CHLl mRNA was strongly expressed by glial cells located in proximal (i.e. retina-near) regions of the optic nerve (Fig. 24b) and low levels of CHL 1 expression were visible in glial cells located in more distal regions of the nerve (Fig. 24b).
  • gilal cells express CHL 1 in vitro
  • cultures of purified astrocytes or oligodendrocytes were prepared from the forebrain of young postnatal mice or rats.
  • the same polyclonal CHLl antibodies which specifically detected CHL l at the cell surface of transfected COS- 1 cells were used.
  • Astrocytes and oligodendrocytes were identified with antibodies to GFAP or with antibodies to the 01 antigen, respectively.
  • Astrocyte cultures contained some cells which were double-labeled by polyclonal CHL 1 (Fig. 25a, d) and monoclonal GFAP (Fig. 25b, e) antibodies.
  • CHL 1 was immunoprecipitated from detergent lysates of whole brain tissue from nine-day-old mice with CHLl antibodies. As control, Ll was similarly immunoprecipitated with polyclonal antibodies from the same brain extract.
  • CHL 1 as another member of the L 1 family of neural recognition molecules found in such diverse species as human, rat, mouse, chicken, zebrafish. and Drosophila. thus constituting a phylogenetically conserved family of molecules all of which are expressed late in development at the onset of axogenesis by neurons and subsets of neurons.
  • L 1 related molecules points to nature's requirement for structurally similar, but functionally most likely distinct neurite outgrowth promoting molecules, and to the evolution of the L 1 family as a group of molecules that may determine the fine- tuning of axonal pathfinding.
  • Drosophila neurogilan a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural cell adhesion molecule Ll . Cell 59:447-460.
  • peripheral myelin glycoprotein Po expresses the L2/HNK-1 and L3 carbohydrate structures shared by neural adhesion molecules. Neurosci. Lett. 82:77-82.
  • Neural cell recognition molecule F 1 1 homology with fibronectin type III and immunoglobulin type C domains. Neuron 2:1351-1361.
  • PANG a gene encoding a neuronal glycoprotein, is ectropically activated by intracisternal A- type particle long terminal repeats in murine plasmacytomas. Proc. Natl. Acad. Sci. (USA) 91:1337-1341.
  • Neural cell adhesion molecule structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science 236:799-806.
  • TAG-1 can mediate homophilic binding, but neurite outgrowth on TAG-1 requires an Ll-like molecule and B, integrins. Neuron 12:675-690.
  • N-CAM neural cell adhesion molecule
  • the axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity.
  • the mouse neuronal cell surface protein F3 A phosphatidylinositol-anchored member of the immunoglobulin superfamily related to chicken contactin. J. Cell Biol. 109:775-788.
  • Transfected F3 F1 1 neuronal cell surface protein mediates intercellular adhesion and promotes neurite outgrowth. Neuron 6:595-606.
  • NEUROMUSCULIN a Drosophila gene expressed in peripheral neuronal precursors and muscles, encodes a cell adhesion molecule. Neuron. 11:673-687. Kayyam, J. F.. Roman. J. M.. de la Rosa. E. J. Schwarz, U. and Dreyer, W. J. (1992). Bravo/NrCAM is closely related to the cell adhesion molecules Ll and Ng ⁇ CAM and has a similar heterodimer structure. J. Cell Biol. 118:1259-1270.
  • Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1. Nature 311:153-155.
  • Ll -mediated axon growth occurs via a hemophilic binding mechanism. Neuron 2:1597-1603.
  • Neural adhesion molecule Ll as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature 334:701-703.
  • MAG myelin-associated glycoprotein
  • Dtrk a Drosophila gene related to the trk family of neurotrophin receptors, encodes a novel class of neural cell adhesion molecule. EMBO J. 111:391-404.
  • Zebrafish neurons express two Ll -related molecules during early axonogenesis. J. Neurosci. Res. 42:547-561.

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