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WO1997040155A1 - Proteines induisant des interactions neuronales/gliales, adn codant pour elles et leurs methodes d'utilisation - Google Patents

Proteines induisant des interactions neuronales/gliales, adn codant pour elles et leurs methodes d'utilisation Download PDF

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Publication number
WO1997040155A1
WO1997040155A1 PCT/US1997/006415 US9706415W WO9740155A1 WO 1997040155 A1 WO1997040155 A1 WO 1997040155A1 US 9706415 W US9706415 W US 9706415W WO 9740155 A1 WO9740155 A1 WO 9740155A1
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Prior art keywords
neuronal
protein
migration
neuronal migration
migration protein
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PCT/US1997/006415
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English (en)
Inventor
Nathaniel Heintz
Mary E. Hatten
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The Rockefeller University
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Publication of WO1997040155A1 publication Critical patent/WO1997040155A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the present invention relates generally to nucleic acid molecules encoding proteins which function in mediating neuron-glia interactions. More specifically, the invention relates to nucleic acid molecules encoding proteins which mediate glial-guided neuronal migration which results in the development of neuronal classes characteristic of cortical regions.
  • this epithelium expands, forming a germinal zone, where postmitotic neuroblasts undergo initial stages of differentiation, and migrate out through the thickening cortical wall to form a series of overlying layers (Sidman and Rakic, 1 973).
  • Rakic (1 971 a, 1 971 b, 1 972) have demonstrated that a system of radial glial fibers provides the primary scaffold for the directed migration of young neurons in cortical regions (Rakic, 1 971 a, 1 971 b, 1 972; Caviness, 1 973; Nowakowski and Rakic, 1 979, 1 981 ; Eckenhoff and Rakic, 1984; Sidman and Rakic, 1 973).
  • the cerebellar cortex has provided an important model for cortical histogenesis, because of its remarkably simple plan and the well-studied migratory pathway of one of the two principal cerebellar neurons, the granule neuron (Rakic, 1 971 b; Rakic and Sidman, 1 973).
  • a protein which functions in guiding neuronal migration along glial cells.
  • the protein contains domains similar to other adhesion and/or signalling molecules.
  • the present invention extends to a neuronal migration (NMP) protein having the following characteristics: a. it guides neuronal migration along glial cells; b. it contains signalling and adhesion domains; c. its mRNA is expressed in cerebrum, cerebellum, hippocampus and olfactory bulb but not in subcortical regions, thalamus or brainstem; and d. it is expressed in zones of immature neurons undergoing axon extension, migration and formation of neuronal layers.
  • NMP neuronal migration
  • the invention relates to antibodies and other binding partners directed to the neuronal migration protein.
  • the neuronal migration protein is termed astrotactin, and has an apparent molecular weight of approximately 1 00-105 kD and has four potential N-linked glycosylation sites
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof encoding a neuronal migration protein; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the neuronal migration protein has a nucleotide sequence or is complementary to a DNA sequence as shown in FIGURE 1 (SEQ ID NO: 1 ).
  • the DNA sequences encoding the neuronal migration protein of the present invention or portions thereof may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the neuronal migration protein.
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector.
  • the present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of or the DNA sequences set forth in FIGURE 1 (SEQ ID NO: 1 ).
  • the preparation of antisense RNA and ribozymes are included herein.
  • the present invention also includes neuronal migration proteins having the activities noted herein, and that display the amino acid sequences set forth and described above and identified by SEQ ID NO:2.
  • the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present neuronal migration protein(s), and more particularly, the complete DNA sequence determined from the sequences set forth above and in SEQ ID NO: 1 .
  • a recombinant expression system is provided to produce biologically active animal or human neuronal migration protein.
  • neuronal migration protein contemplates that specific molecules exist for guiding the migration of cells during development, such as neurons, as described earlier. Accordingly, the exact structure of each neuronal migration protein will understandably vary so as to achieve the appropriate adhesion and signalling activity specificity. It is this specificity and the direct involvement of the neuronal migration protein in the guiding cells to their ultimate destination which offers the promise of a broad spectrum of diagnostic and therapeutic utilities.
  • the present invention naturally contemplates several means for preparation of the neuronal migration protein, including, as illustrated herein, known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope.
  • the isolation of the cDNA and amino acid sequences disclosed herein facilitates the reproduction of the neuronal migration protein by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
  • the invention includes an assay system for screening of potential drugs effective to modulate the adhesion and/or signalling activity of the neuronal migration protein by interrupting or potentiating the interaction of neurons and glia, such interaction including adhesion and other signalling mechanisms.
  • the test drug could be administered to a cellular sample with a molecule that modulates the adhesion and/or signalling capacity of the neuronal migration protein, or an extract containing the neuronal migration protein, to determine its effect upon the interaction of neurons and glia to one another or to any chemical sample, or to the test drug, by comparison with a control.
  • the assay system can more importantly be adapted to identify drugs or other entities that are capable of binding to the neuronal migration protein, thereby inhibiting or potentiating adhesion or signalling activity.
  • Such an assay would be useful in the development of drugs that would be specific against particular migratory activity, or that would potentiate such activity, in time, or in level of activity.
  • drugs might be used to modulate migration of cells to areas of damaged tissue, or to treat other pathologies, as for example, in treating diseases such as those of the nervous system, including, but not limited to Zellweger syndrome, childhood epilepsy, Schizophrenia, pediatric tumors, microencephaly and cerebellar hyperplasia.
  • the invention contemplates antagonists of the activity of a NMP, in particular, an agent or molecule that inhibits NMP activity.
  • the antagonist can be a peptide having the sequence of a portion of a signalling or adhesion domain of a NMP.
  • the diagnostic utility of the present invention extends to the use of the present NMP in assays to screen for disorders associated with altered cellular migration, and for molecules which modulate that migration.
  • the present invention likewise extends to the development of antibodies against the NMPs, 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 NMPs of alternate species, or that encode related proteins.
  • 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 NMP activity.
  • the NMPs, 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 NMP that has been labeled by either radioactive addition, or radioiodination.
  • immunoassays such as a radioimmunoassay
  • 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.
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 CI, 5, Cr, "Co, 58 Co, 59 Fe, 90 Y, 125 l, ,31 l, and 186 Re
  • known currently available counting procedures may be utilized
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotomet ⁇ c, amperometnc or gasomet ⁇ c 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 NMP, 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 NMPs, 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 based upon the activity of the NMPs, 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 prevention of the manifestations of conditions such as those recited earlier herein, that are or may be causally related to or following from the altered binding activity or signalling activity of the NMP or its subunits, and comprises administering an agent capable of modulating the production and/or activity of the NMP or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.
  • the therapeutic method generally referred to herein includes a method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions which comprise effective inhibitors or enhancers of activation of the NMP 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 which comprise effective inhibitors or enhancers of activation of the NMP 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 NMP or proteins, as represented by SEQ ID NO:2 can be administered to inhibit or potentiate NMP activity.
  • proteins of the present invention whose sequences are presented in SEQ ID NOS:2 herein, its antibodies, agonists, antagonists, or active fragments thereof, can be prepared in pharmaceutical formulations for administration in instances wherein therapy for inducing cellular migration is appropriate, such as to treat diseases including those of the nervous system.
  • compositions for use in therapeutic methods which comprise, or are based upon, the NMP, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic, potentiate or antagonize the activities of the NMP.
  • FIGURE 1 DNA sequence of GC14
  • FIGURE 2 Structural motifs of astrotactin
  • A Schematic representation of the domain structure of astrotactin.
  • the filled box represents the signal peptide.
  • the shaded boxes represent EGF repeats.
  • the hatched boxes represent Fnlll repeats.
  • B Alignment of the three EGF repeats and the two Fnlll repeats in astrotactin over the consensus of the respective motifs. The identical amino acids are shown in bold. The underlined amino acids are substitutes found in Fnlll repeats of other proteins at the same positions. Dashes denote gaps to optimize the alignment.
  • ASTFN astrotactin
  • FIGURE 3 Northern Analysis of GC14 mRNA Expression
  • RNA or poly(A) + RNA was isolated from various mouse tissues or purified primary cells, separated on 1 % agarose-formaldehyde gels, and blotted to nylon membranes. The blots were hybridized with a cDNA probe derived from the astrotactin 3' non-coding region. As a loading control, the same blots were hybridized with GAPDH probe.
  • A In early postnatal animals astrotactin is expressed in forebrain and cerebellum F, forebrain; H, heart; K, kidney; C, cerebellum; Li, liver; Lu, lung; S, spleen; T, thymus.
  • B Astrotactin mRNA is detected in purified primary neurons but not in glia. N, granule cells; G, glial cells
  • C Astrotactin is expressed at high levels during cerebellum development. E, embryonic days; P, postnatal days; A, adult.
  • FIGURE 4 Localization of GC14 mRNA by in situ hybridization.
  • the astrotactin mRNA is localized in parasagittal sections of postnatal day 10 mouse brain by in situ hybridization with a digoxigenin-labeled antisense probe.
  • a and C In the cerebellum, labeled cells are detected in the inner layer of EGL, ML and IGL. At higher magnification (C), proliferating precursor cells in the superficial zone of the EGL (ELa) are not labeled.
  • B and D In developing hippocampal formation, strong expression of astrotactin mRNA is present in the granule cells of dentate gyrus (DG) At high magnification (D), proliferating cells in the subgranular layer (SGLa) are unlabeled.
  • E In developing cortex, newly generated cells of layer 2/3 are most intensely labeled, with fewer labeled cells in deeper layers.
  • F In olfactory bulb, expression of astrotactin mRNA is evident in postmitotic cells in the GL, MCL and GCL.
  • EGL external germinal cell layer
  • ML molecular layer
  • IGL internal granule cell layer
  • CA cornis ammoms
  • DG dentate gyrus
  • SGL subgranular layer
  • GL glomerular layer
  • MCL mitral cell layer
  • GCL granule cell layer.
  • the scale bar represents 50 ⁇ m in (A,B,E,F) and 1 2.5 ⁇ m in (C and D).
  • FIGURE 5 Immunoblot of GC 14 protein in mouse cerebellum.
  • Triton X-100 extracts from postnatal day 6 mouse cerebellum were resolved on 10% SDS-polyacrylamide gels, blotted and stained with antisera at 1 : 1000 dilution.
  • A Staining with preimmune serum.
  • B Staining with the affinity purified ant ⁇ -GC14 antibody, which recognizes a protein of about 100 kD (arrow).
  • C Staining with the depleted ant ⁇ -GC1 4 antiserum at 1 : 100 dilution,
  • FIGURE 6 Immunocytochemical Localization of astrotactin protein in developing mouse cerebellum (P6)
  • FIGURE 7 Astrotactin mediates neuron-glia interactions in vitro
  • Enriched granule cell/ghal cell cultures were prepared as described in the methods, and plated at 0.5 x 10 6 cells/ml, in the absence or presence of antisera. After 48 hour in vitro, the cultures were stained with anti-GFAP antibody in order to visualize glial processes and measure the distance of unstained granule cells from these processes (Edmondson, et al, 1988).
  • A In the absence of ant ⁇ -GC 1 4 antibody, astroglia extend long, thin processes. Unstained neurons align along stained glial fibers.
  • B Similar results were obtained with the "flow-though" fraction collected after affinity purification of ant ⁇ -GC14 antibody on a GST-GC14 fusion protein column.
  • FIGURE 8 Quantitation of inhibition of Cerebellar Neuron-glia Interactions by Antibodies raised against GC14 Protein The percentage of inhibition, as compared to the control in each cell culture experiment is plotted in the left panel. As a control (right panel), we counted the total number of neurons and glia after treatment with antibodies. Control, in the absence of antibodies; Dplt, in the presence of depleted anti- GC14 Fab fragments; GC14; in the presence of affinity-purified ant ⁇ -GC14 Fab fragments; ASTN; in the presence of the eluted astrotactin antibodies from GST- GC14 fusion protein column.
  • FIGURE 9 Expression of Astrotactin During Cerebellar Granule Cell Development
  • Proliferating precursor cells ( 1 ), occupying the superficial aspect of the EGL, do not express astrotactin mRNA or protein. Rather expression commences (filled cells) in the deeper aspect of the EGL where postmitotic precursor cells bind to astroglial fibers (BF), extend parallel fibers (2), migrate along the Bergmann glial fibers (b) (3-5), detach from the glial fibers and move past Purkinje neurons (P) (6) with a T-shaped parallel fiber axon trailing behind.
  • BF astroglial fibers
  • b extend parallel fibers
  • Purkinje neurons P
  • neuron-glia interaction protein involved in neuron-glia interaction
  • neuron-glia interaction protein involved in neuron-glia interaction
  • 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 or its named subunits.
  • neuron-glia ⁇ nteract ⁇ on(s) neuron-glia ⁇ nteract ⁇ on(s)
  • neuron-glia ⁇ nteract ⁇ on(s) protein involved in neuron-glia ⁇ nteract ⁇ on(s)
  • neuron-glia ⁇ nteract ⁇ on(s) protein involved in neuron-glia ⁇ nteract ⁇ on(s)
  • ammo 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 2 refers to the free ammo group present at the ammo 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 whpse 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 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 deoxy ⁇ bonucleotides (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 nontranscnbed 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' (ammo) 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.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with nuclease S1 ), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain
  • 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 the probe of the present invention, 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 1 5-25 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 being described in further detail in U.S. Patent Nos. 4,81 6,397 and 4,81 6,567.
  • 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 intact immunoglobulin molecules, and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • Fab and F(ab') 2 portions of antibody molecules are prepared by the proteolytic 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') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and 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 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.
  • phrases “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a mammal, and in particular, 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 5 x SSC and 65 °C for both hybridization and wash.
  • the present invention concerns the identification of a cDNA encoding a protein which plays a role in guiding the migration of cells.
  • this protein guides neuron migration by mediating neuron-glia interaction.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a NMP, or a fragment thereof, that possesses a molecular weight of about 100 kD and an amino acid sequence as set forth in FIGURE (SEQ ID NO:2); preferably a nucleic acid molecule, in particular, a recombinant DNA molecule or cloned gene, encoding the 100 kD NMP has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 1 (SEQ ID NO: 1 ) .
  • the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the NMP is implicated, to modulate the activity initiated by the NMP.
  • an appropriate inhibitor of the NMP can be introduced to block the interaction of the NMP with those factors causally connected with such migration.
  • instances where insufficient migration is taking place could be remedied by the introduction of additional quantities of the NMP or its chemical or pharmaceutical cognates, analogs, fragments and the like.
  • the NMPs or their binding partners or other ligands or agents exhibiting either mimicry or antagonism to the NMP or control over their production may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with altered cellular migration for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the NMPs or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the NMPs and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions associated with altered cellular migration patterns.
  • the NMP or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • small molecules that mimic or antagonize the activity(ies) of the NMP of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M.
  • Panels of monoclonal antibodies produced against NMP peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc.
  • monoclonal antibodies that neutralize the activity of the NMP or its subunits can be readily identified in in vitro assays as described below.
  • High affinity antibodies are also useful when immunoaffinity purification of native or recombinant NMP is possible.
  • the anti-NMP antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti-NMP antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions of whole antibody molecules.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a NMP/protein, such as an anti-NMP antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • the anti-NMP antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions or whole antibody molecules.
  • patients capable of benefiting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection or other like pathological derangement.
  • Methods for isolating the NMP and inducing anti-NMP antibodies and for determining and optimizing the ability of anti-NMP antibodies to assist in the examination of the target cells are all well-known in the art.
  • a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an adhesion or signalling portion thereof.
  • Splenocytes are typically fused with myeloma cells using polyethylene glycol (PEG) 6000.
  • Fused hybrids are selected by their sensitivity to HAT.
  • Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present NMP and their ability to inhibit specified NMP activity in target cells.
  • a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
  • the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
  • the antibody-containing medium is then collected.
  • the antibody molecules can then be further isolated by well-known techniques.
  • DMEM Dulbecco's minimal essential medium
  • fetal calf serum An exemplary inbred mouse strain is the Balb/c.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a NMP, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • the composition comprises an antigen capable of modulating the specific binding of the present NMP within a target cell.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of NMP activity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • the therapeutic compositions may further include an effective amount of the NMP/NMP antagonist or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid.
  • active ingredients an antibiotic, a steroid.
  • Intravenous Formulation I Ingredient mg/ml cefotaxime 250.0
  • Intravenous Formulation II Ingredient mg/ml ampicillin 250.0 NMP 1 0.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1 .0 ml PC17US97/06415
  • Intravenous Formulation IV Ingredient mg/ml NMP 10.0 dextrose USP 45.0 sodium bisulfite USP 3.2 edetate disodium USP 0.1 water for injection q.s.a.d. 1 .0 ml
  • Intravenous Formulation V Ingredient mg/ml NMP antagonist 5.0 sodium bisulfite USP 3.2 disodium edetate USP 0.1 water for injection q.s.a.d. 1 .0 ml
  • pg means picogram
  • ng means nanogram
  • ug means microgram
  • mg means milligram
  • ul means microliter
  • ml means milliliter
  • I means liter.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • phage DNAS e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g., M 1 3 and filamentous single stranded
  • any of a wide variety of expression control sequences - sequences that control the expression of a DNA sequence operatively linked to it -- may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TA C system, the TRC system, the L TR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast ⁇ -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1 , COS 7, BSC 1 , BSC40, and BMT10), insect cells (e.g. , Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1 , COS 7, B
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • NMP analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention.
  • Analogs, such as fragments may be produced, for example, by pepsin digestion of NMP material.
  • Other analogs, such as muteins can be produced by standard site-directed mutagenesis of NMP coding sequences.
  • Analogs exhibiting "NMP activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding NMP can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the NMP ammo acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 ( 1 981 ); Nambair et al., Science, 223: 1 299 ( 1984); Jay et al., J. Biol. Chem. , 255:631 1 ( 1 984)
  • DNA sequences allow convenient construction of genes which will express NMP analogs or "muteins".
  • DNA encoding muteins can be made by site-directed mutagenesis of native NMP genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention extends to the preparation of antisense oligonucleotides and ribozymes that may be used to interfere with the expression of the NMP at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule. (See Weintraub, 1 990; Marcus-Sekura, 1 988.) In the cell, they hybridize to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, since they are easy to synthesize and are likely to pose fewer problems than larger molecules when introducing them into NMP-producmg cells. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura, 1 988; Hambor et al., 1 988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these RNAs, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1 988.). Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type Tetrahymena-type and "hammerhead”-type.
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead”-type recognize eleven- to eighteen-base sequences.
  • the DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for NMPs and their ligands.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present NMP.
  • the NMP can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular NMP activity in suspect target cells.
  • ant ⁇ body( ⁇ es) to the NMP can be produced and isolated by standard methods including the well known hybridoma techniques.
  • Ab the ant ⁇ body( ⁇ es) raised in another species as Ab 2
  • NMP neurotrophic factor
  • a number of useful procedures are known. Three such procedures which are especially useful utilize either the NMP labeled with a detectable label, antibody Ab, labeled with a detectable label, or antibody Ab 2 labeled with a detectable label.
  • the procedures may be summarized by the following equations wherein the asterisk indicates that the particle is labeled, and "NMP" stands for the neural migration protein:
  • NMP* + Ab, NMP*Ab
  • the NMP forms complexes with one or more ant ⁇ body( ⁇ es) or binding partners and one member of the complex is labeled with a detectable label.
  • a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • Ab 2 will react with Ab, .
  • Ab raised in one mammalian species has been used in another species as an antigen to raise the antibody Ab 2 .
  • Ab 2 may be raised in goats using rabbit antibodies as antigens.
  • Ab 2 therefore would be anti-rabbit antibody raised in goats.
  • Ab will be referred to as a primary or anti-NMP antibody, and Ab 2 will be referred to as a secondary or anti-Ab, antibody.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the NMP or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 32 P, 35 S, 36 CI, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 l, 13, l, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotomet ⁇ c, amperomet ⁇ c or gasomet ⁇ c techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodnmides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,01 6,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined NMP activity or predetermined NMP activity capability in suspected target cells.
  • one class of such kits will contain at least the labeled NMP or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive, " "sandwich, " "DASP' and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence or capability of cells for predetermined NMP activity, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g “competitive,” “sandwich,” “double antibody,” etc.), and comprises:
  • an assay system for screening potential drugs effective to modulate the activity of the NMP may be prepared.
  • the NMP may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the migration activity of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known NMP.
  • assays were used to identify an immune activity, astrotactin (Edmondson et al, 1 988), that functions in neuron-ghal interaction and in neuronal migration (Stitt and Hatten, 1 990; Fishell and Hatten, 1 991 ).
  • This polyclonal astrotactin antibodies recognize a major band of M r 100, which by Western Blot analysis and immunocytochemical localization, is expressed by migrating neurons in development cerebral and cerebellar cortex (Stitt et al, 1 990).
  • the spatiotemporal pattern of expression of astrotactin is unique among cell surface receptor systems described to date, as it is only expressed by CNS neurons undergoing glial-guided neuronal migration and assembly into neuronal layers.
  • the molecular cloning of astrotactin has been achieved as part of a broad screen for cDNAs that mark specific stages of cerebellar, granule cell development, including proliferation, axon extension, migration and terminal differentiation.
  • This screen yielded a set of 39 novel clones that are differentially regulated at the transcriptional level during cerebellar development (Kuhar et al, 1993)
  • the complete ammo acid sequence of GC 14 one of these clones is reported, and it is demonstrated that antibodies against a bacte ⁇ ally expressed GC 14 fusion protein recognize a cerebellar, neuronal protein with M r 100 by Western blot analysis.
  • This protein is expressed in cortical regions of the CNS (cerebral cortex, cerebellar cortex, hippocampus, olfactory bulb) in differentiating and migrating neurons.
  • Antibodies raised against the GC14 fusion protein mimic the blocking activity of anti-astrotactm antibodies in functional assays of neuron-glial interactions (Edmondson et al., 1 988), thus in vitro functional assays establish that GC14 encodes the astrotactin activity.
  • a comparison of the deduced ammo acid sequence of astrotactin reveals that astrotactin encodes a novel protein containing interspersed EGF and fibronectin type III motifs, suggesting a dual role in neuron-glial adhesion and cell-cell signaling.
  • cDNAs encoding the 5' terminus of the astrotactin protein a randomly primed, granule neuron cDNA library was constructed in ⁇ ZAPII vector from 8 x 10 8 granule cells purified from P5 mouse cerebellum (Hatten, 1 985; Gao et al, 1 993; Qian and Hatten, unpublished). The 5' GC1 4 cDNA fragment was used to screen this library.
  • GC14 cDNAs were also isolated from a mouse brain oligo dT ⁇ ZAPII cDNA library, generously provided by Dr. J. Friedman (Rockefeller University) and a mouse cerebellar oligo dT ⁇ ZAPII cDNA library (Dietz and Hatten, unpublished).
  • GC14 cDNA inserts were excised directly as Bluescript plasmids (Stratagene) and the nucleotide sequence of the inserts analyzed by dideoxy chain termination sequencing methods (Sanger et al, 1 977). Nucleotide sequence information was checked against existing GenBanks to determine homology with known proteins.
  • Anti-sense and sense RNA probes against calbindin a marker for Purkinje neurons were used as positive controls.
  • a cDNA fragment containing the extracellular domain covering the region of the EGF and Fnlll repeat (SEE BOX IN FIGURE 2a), was subcloned into the PGEM-3X vector (Pharmacia), introduced into E. coli DH5 ⁇ cells by transformation.
  • Expressed GST-fusion protein was purified in native form by cell lysis followed by centrifugation to generate the endosomal fraction, and affinity chromatography using glutathione-Sepharose 4B column to obtain pure protein. Methods were as described by the manufacturer. Polyclonal antisera were raised in rabbits.
  • Vibratome sections of C57B1 /6J murine brain were prepared. After pre-incubation with normal goat serum (Gibco, 10%, 20°C, 1 hour), ant ⁇ -GC 14 antiserum was applied to the sections (1 :200) overnight (4°C). FITC conjugated anti-rabbit secondary antibodies were used at 1 : 100 (Sigma). Photomicrospy was as described for in situ hybridization.
  • Quantitation of the distance of neurons from GFAP-positive glial fibers was performed as described (Edmondson et al, 1 988), with the following modifications.
  • Cells were imaged using a Hamamatsu intensified charge-coupled device (l-CCD) 2400-50/80 camera head and controller mounted on a Zeiss Axiovert-35 inverted microscope equipped with differential interference contrast optics (20X/0.5, 40X/0.75, 63X/1 .4, or 1 00X/1 .3 NA objectives). Images were recorded with a Panasonic optical memory disk recorder and measurements of neuron-glia distances or of glial process length were made with an Metamorph Image Analysis System (Universal Imaging).
  • l-CCD Hamamatsu intensified charge-coupled device
  • a set of candidate clones was generated as described previously by screening a cDNA expression library, construed from purified p3-p5 granule neurons, with a polyclonal anti-astrotactin antiserum (Kuhar et al, 1993).
  • a polyclonal anti-astrotactin antiserum As the anti-astrotactin antiserum (Stitt and Hatten, 1 990) recognizes a CNS-specific antigen with an apparent M, 100 kD, brain-specific cDNAs that hybridize to mRNAs larger than 3 kb were considered as candidate clones.
  • GC14 encoded a brain-specific transcript of approximately 7 kb with a temporal and spatial pattern of expression similar to that of the astrotactin antibody. Therefore, GC14 was characterized in detail, and a series of cDNAs isolated to obtain primary sequence information (FIGURE 1 ). Of twenty-one cDNA clones isolated from a randomly primed cDNA library prepared from purified granule cells (P5), eight encoded overlapping sequences that covered 5.4 kb of the approximately 7 kb GC14 mRNA.
  • GC14 Encodes a Novel Neuronal Protein Containing both Fibronectin Type III and EGF Repeats
  • GC14 encodes a 100-105 kD polypeptide with four potential N-linked glycosylation sites (FIGURE 1 ).
  • the N-terminus of GC14 begins with a canonical signal peptide 1 5-1 6 amino acids in length (FIGURE 2).
  • FIGURE 2 Within the extracellular domain, the most notable feature of the deduced GC14 protein is the presence of repeated domains homologous to other well characterized adhesion or signaling molecules (FIGURE 2).
  • the sequence of the extracellular domain of GC14 contains 3 cysteine-rich repeats with a high degree of homology to the epidermal growth factor (EGF), consisting of a stretch of approximately 40 amino acids in which cysteine is highly conserved.
  • EGF epidermal growth factor
  • cysteine-rich motifs occur in the extracellular domains of the EGF receptor, and are also found in several other well-characterized signaling molecules, including TAN-1 (Ellisen et al, 1 991 ), lin- 12 (Greenwald, 1 994) and the Drosophila neurogenic genes Notch, Delta, and Serrate (Greenwald, 1 994; Vassin et al, 1 987; Thomas et al, 1 991 ).
  • GC14 Interspersed with the EGF repeats in GC14 is a second domain of homology with molecules that function in neural cell adhesion. As shown in FIGURE 2B, GC14 contains two repeats with homology to a 90 amino acid motif found in fibronectin, Fnlll (Skorstengaard et al, 1 986).
  • GC14 hybridized to a single mRNA band of approximately 7 kb, expressed in brain, but not kidney, heart, liver, lung, thymus or spleen (FIGURE 3A).
  • the 7 kb GC14 mRNA band was detected in cortical regions, including cerebellum and forebrain (FIGURE 3A). Transcripts were not detected in subcortical regions, thalamus or brainstem (not shown).
  • GC1 4 mRNA was expressed in purified cerebellar granule cells, but not glial cells (FIGURE 3b).
  • GC 14 encoded a brain-specific mRNA expressed in development neurons.
  • GC1 4 transcripts were abundant in cortical regions of development murine brain including neocortex, cerebellum, hippocampus, and olfactory bulb.
  • P0-P10 neonatal cerebellar cortex
  • labeled cells were abundant in the deeper aspect of external germinal layer, where cells are undergoing early steps in differentiation, and in the internal granule cell layer, where postmigratory neurons are forming glomeruli (FIGURE 4A).
  • Glial cells including the Bergmann glia of the molecular layer and astrocytes within the white matter were unlabeled.
  • FIGURE 4B A similar pattern of expression was seen in the development hippocampal formation (FIGURE 4B), where heavily labeled cells were observed in the subgranular layer of the immature dentate gyrus (Eckenhoff and Rakic, 1 984).
  • Overlying cortex newly generated cells of layers 2/3 were intensely labeled, with scattered labeled cells seen within the deeper layers (FIGURE 4E); labeled cells were not detected in the collapsing ventricular zone, the major germinal zone of cortex (now shown).
  • Transcripts were also abundant in the developing olfactory bulb, where postmitotic, immature neurons within the granule cell layer, mitral cell layer and glomeruli were labeled (FIGURE 4F).
  • GC14 mRNA was not detected in the deep nuclei of the cerebellar cortex, subcortical brain regions, thalamus, midbrain or brainstem (not shown).
  • astroglial cells purified from early postnatal cerebellum were treated with antibodies against expressed GC14 fusion protein. No effects were seen on cerebellar astroglial cell survival, proliferation or differentiation.
  • removal of the anti-GC14 antibodies, by washing 4 hours after plating resulted in elaborate glial process formation and glial organization of neuronal positioning (data not shown).
  • astrotactin has been deduced from GC14, a neuron specific cDNA, isolated in a broad screen of genes that mark specific stages of cerebellar granule cell development.
  • functional assays demonstrate that GC14 encodes the neural antigen astrotactin, previously shown to function in migration along the glial fiber system (Fishell and Hatten, 1 991 ) and positioning of postmigratory neurons along glial fibers in vitro (Edmondson et al, 1 988) .
  • the temporo-spatial pattern expression of astrotactin, in regions of brain that form layered structures via directed cell migrations, suggests that this gene functions in the development of neuronal classes characteristic of cortical regions.
  • the cloning of astrotactin provides the first opportunity to examine the molecular basis of cell signaling involved in glial-guided neuronal migration in brain.
  • astrotactin The deduced structure of astrotactin reveals a novel class of cell surface receptor system that shares structural motifs with the EGF receptor and the fibronectin gene family. This finding, together with the spatially restricted expression of astrotactin, in newly generated CNS neurons, raises the possibility that astrotactin may provide a signaling system that induces aspects of the development of those regions of CNS in which neurons utilize the glial scaffolding system for cell positioning. These regions include cerebrum, cerebellum, hippocampus, and olfactory bulb (Rakic, 1 990). The relative contributions of astrotactin to the signaling required for the specification of neuronal identity, via a particular molecular program of differentiation and interaction with astroglial fibers per se remains to be determined.
  • the domain structure of astrotactin suggests that the protein provides a unique cell surface receptor system, containing domains canonical for both signaling and adhesion functions.
  • Comparison of the predicted protein sequence of astrotactin revealed a high degree of sequence homology with two classes of molecules, the EGF receptor family and neural adhesion molecules of the fibronectin family.
  • the cysteine-rich EGF repeats are found in several other well- characterized signaling molecules, including TAN- 1 , line- 12 and the Drosophila neurogenic genes Notch, Delta, and Serrate (Ellisen et al, 1 991 ; Greenwald, 1 994; Vassin et al, 1 987; Thomas et al, 1 991 ).
  • Fnlll domain a domain proposed to function in cell-cell adhesion.
  • L1 structure/function studies have demonstrated that Fnlll repeats are sufficient for cerebellar neuron adhesion (Appel et al., 1 993).
  • receptor systems containing Fnlll motifs are common in axonal glycoproteins that are members of the immunoglobulin gene superfamily, including L1 , TAG-1 , contactin, fasciclin II, and neuroglian.
  • a set of IgG repeats of the C2 type are followed by a series of Fnlll repeats.
  • EGF and Fnlll repeats While the function of EGF and Fnlll repeats has been analyzed in other molecules, their arrangement in the deduced structure of astrotactin is quite different. This may therefore point to a novel mechanism of action of these domains within astrotactin.
  • the identification of the astrotactin gene allows for site-directed mutagenesis studies of astrotactin to dissect the function of specific domains of the protein. In particular, it may be determined whether disruption of EGF domains interferes with cell differentiation processes, with disruption of the Fnlll domain effecting adhesion mechanisms.
  • cortical regions are the only areas where young neurons use the glial system as a scaffold for migration.
  • astrotactin functions in the process of neuronal migration along glial fibers.
  • Astrotactin was not expressed in subcortical regions of brain, thalamus, midbrain or brainstem, regions where young neurons utilize a "neurophilic" mode of migration, ignoring the glial fibers and assembling into ganglionic rather than laminar structures (Rakic, 1978, 1 990).
  • astrotactin appears to provide a means for newly generated neuronal precursors to use the glial scaffold as a guidance system for migration.
  • molecules such as astrotactin would have evolved during the period when laminar structures appeared.
  • the specialized, laminar architecture of vertebrate cortex would provide a framework to facilitate numerous features of synaptic connectivity unique to cortex, including the input-output circuitry (Gilbert, 1 983, 1 992), interlaminar circuitry between specific layers and the development of horizontal connections between distant visuotopic or somatopic loci in cortex (Gilbert, 1 993).
  • the astrotactin gene has been shown to be highly conserved across vertebrate species, including human, monkey, rat, mouse, and Xenopus. Homologous genes were not detected in lower animals (C.
  • Astrotactin expression commences within the deeper aspect of the EGL, where postmitotic EGL precursor cells intermingle with the radially arrayed fibers of the Bergmann glia (Ramon y Cajal, 1 889; Fujita, 1 967; Rakic and Sidman, 1 972).
  • Gao et al As studies by Gao et al (1 991 ) demonstrate that neuron-glia binding can inhibit EGL cell proliferation, the expression of astrotactin may promote the exit of proliferating precursors cells from the cell cycle and entry into a neuronal differentiation pathway (Gao et al, 1 991 ). Thereafter, neuron-glia migration via the astrotactin receptor system would signal or maintain a program of changing gene expression shown to constitute granule cell development (Kuhar et al, 1 993). Migration may serve to drive the young neurons through a temporospatial matrix of extracellular cues required for the elaborate program of granule cell differentiation. In this model, genes such as astrotactin would provide a molecular basis for neuron-glia binding and for subsequent steps in migration and terminal differentiation of the granule cell.
  • astrotactin The functional role of astrotactin in neuron-glial interactions in cortical regions of the developing brain is supported by the pattern of expression of the astrotactin protein. Immunocytochemical localization of antibodies against recombinant astrotactin protein reveals that astrotactin is expressed by newly generated neuronal precursor cells. The level of astrotactin expression by postmigratory cells is temporally regulated, as the mRNA transcription level falls 5-1 0 fold within a week of granule neuron assembly into the EGL. The continued expression of astrotactin within adult cerebellar cortex possibly reflects the elaboration of other kinds of neuron-glial contacts seen in mature brain, such as the enwrapping of synaptic structures by glial cells.
  • velate astroglial cells envelop glomeruli formed by granule neurons and their afferent axons, the mossy fibers.
  • small clusters of granule cells extend short, radiating dendrites which converge upon mossy fiber terminals (Ramon y Cajal, 1 91 1 ).
  • the granule cell dendrites, disposed on the surface of the mossy fiber rosette, are subsequently encapsulated by velate astroglial cells (Palay and Chan-Palay, 1 974).
  • genes such as astrotactin would mediate neuron-glia signaling events responsible for cellular differentiation within cortical laminae.
  • astrotactin functions in the movement of the neuron along the glial guide, this model would argue that the interactions between the young neuron and the glia scaffold would provide a permissive environment for the expression of genes needed to specify those classes of neuronal cells found in neuronal layers of vertebrate cortical regions.
  • Neural cell adhesion molecule structure, immunoglobulin-like domains, cell surface modulation, and alternative
  • Astrotactin a novel cell surface antigen that mediates neuron-glia interactions in cerebellar microcultures. J. Cell Biol. 106, 505-51 7. Ellisen, L., Bird, J., West, D.C, Soreng, A.L., Reynolds, T.C, Smith, S.D., and Sklar, J. (1 991 ). Tan-1 , the human homolog of the Drosophila Notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms. Cell 66, 649-661 .
  • the axonal glycoprotein TAG-1 is an immunoglobulin superfamily member with neurite outgrowth-promoting activity.
  • Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro. Neuron 6, 705- 71 5.
  • Developmentally regulated cDNAs define four stages of cerebellar granule neuron differentiation. Development 1 1 2, 97-104.
  • Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature 334, 701 -703.
  • Rakic, P. ( 1 971 b). Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electron microscopic study in
  • Rakic, P. ( 1 972). Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol. 145, 61 -84.
  • the Drosophila Serrate encodes an EGF-like transmembrane protein with a complex expression pattern in embryos and wing discs. Development 1 1 1 , 749-761 .
  • the neurogenic gene Delta of D. Melanogaster is expressed in neurogenic territories and encodes a putative transmembrane protein with EGF-like repeats. EMBO, J. 6, 3431 -3440.

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Abstract

L'invention porte sur un antigène neuronal du SNC qui intervient dans les interactions neurone/glie qui sont la clef du développement du SNC cérébral, y compris la migration guidée par le glial. Cet antigène agit en tant que nouvelle molécule signal exprimée par des neurones récemment créés dans le cerveau en développement et dont la séquence d'acides aminés déduite fait apparaître une nouvelle structure secondaire comportant trois facteurs de croissance de l'épithélium et deux répétitions de fibronectine de type III. L'invention porte également sur les acides nucléiques codant pour l'antigène neuronal, sur les anticorps agissant contre lesdits antigènes, et sur des acides nucléiques antisens et des ribozymes agissant contre les acides nucléiques. L'invention porte en outre sur des équipements de diagnostic et des compositions thérapeutiques et les procédés associés pouvant comprendre ou être dérivés desdits acides nucléiques, et l'antigène, ainsi que les anticorps, les molécules antisens et les ribozymes dirigés contre eux.
PCT/US1997/006415 1996-04-19 1997-04-17 Proteines induisant des interactions neuronales/gliales, adn codant pour elles et leurs methodes d'utilisation WO1997040155A1 (fr)

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US08/635,061 1996-04-19

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038576A3 (fr) * 1999-11-24 2002-07-11 Whitehead Biomedical Inst Polymorphismes humains a nucleotide unique
WO2020041532A1 (fr) * 2018-08-21 2020-02-27 Sf17 Therapeutics, Inc. Compositions et méthodes pour traiter des maladies neurodégénératives progressives

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Title
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EDMONSON J. ET AL.: "Astrotactin: A novel neuronal cell surface antigen that mediates neuron-astroglial interactions in cerebellar microcultures", THE JOURNAL OF CELL BIOLOGY, vol. 106, February 1988 (1988-02-01), pages 505 - 517, XP002037215 *
FINK J. ET AL.: "The CNS neuronal migration gene, astrotactin, is mapped to human chromosome band 1q25 by fluorescence in situ hybridization", AMERICAN JOURNAL OF HUMAN GENETICS, vol. 57, no. 4, 1995, pages 750, XP002037219 *
HATTEN M.: "The role of migration in central nervous system neuronal development", CURRENT OPINION IN NEUROBIOLOGY, vol. 3, 1993, pages 38 - 44, XP002037221 *
HILLIER L. ET AL.: "The WashU-Merck EST project, AC R59057", EMBL DATABASE, 29 May 1995 (1995-05-29), HEIDELBERG, XP002037213 *
KUHAR S. ET AL.: "Changing patterns of gene expression define four stages of cerebellar granule neuron differentiation", DEVELOPMENT, vol. 117, 1993, pages 97 - 104, XP002037217 *
ROSS M. ET AL.: "Cloning of the neuron-glia ligand astrotactin using cDNA libraries from early postnatal granule neurons", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 16, no. 1, 1990, pages 68.6, XP002037214 *
STITT T. AND HATTEN M.: "Antibodies that recognize astrotactin block granule neuron binding to astroglia", NEURON, vol. 5, November 1990 (1990-11-01), pages 639 - 649, XP002037216 *
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ZHENG C. ET AL.: "CNS gene encoding astrotactin, which supports neuronal migration along glial fibers", SCIENCE, vol. 272, 19 April 1996 (1996-04-19), pages 417 - 419, XP002037222 *
ZHENG C. ET AL.: "Molecular cloning of astrotactin", SOCIETY FOR NEUROSCIENCE ABSTRACTS, vol. 20, no. 1-2, 1994, pages 682.4, XP002037218 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001038576A3 (fr) * 1999-11-24 2002-07-11 Whitehead Biomedical Inst Polymorphismes humains a nucleotide unique
WO2020041532A1 (fr) * 2018-08-21 2020-02-27 Sf17 Therapeutics, Inc. Compositions et méthodes pour traiter des maladies neurodégénératives progressives
US11396542B2 (en) 2018-08-21 2022-07-26 Synkrino Biotherapeutics, Inc. Astrotactin1-based compositions and pharmaceutical formulations

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