WO1994004562A1 - Compositions concernant gap-43 des mammiferes et procedes d'utilisation - Google Patents

Compositions concernant gap-43 des mammiferes et procedes d'utilisation Download PDF

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WO1994004562A1
WO1994004562A1 PCT/US1993/007643 US9307643W WO9404562A1 WO 1994004562 A1 WO1994004562 A1 WO 1994004562A1 US 9307643 W US9307643 W US 9307643W WO 9404562 A1 WO9404562 A1 WO 9404562A1
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gap
cells
protein
cell
peptide
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WO1994004562A9 (fr
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Mark C. Fishman
Howard Federoff
Mauricio Zuber
Stephen Strittmatter
Dario Valenzuela
Shi-Chung Ng
Edward Grabczyk
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The General Hospital Corporation
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10Tetrapeptides
    • C07K5/1002Tetrapeptides with the first amino acid being neutral
    • C07K5/1005Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702Regulators; Modulating activity
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00Nucleic acids vectors
    • C12N2800/10Plasmid DNA
    • C12N2800/108Plasmid DNA episomal vectors
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/008Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/80Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to the fields of molecular genetics and
  • the invention relates to the cDNA sequence and corresponding amino acid sequence of mammalian GAP-43, a neuronal
  • the present invention is further related to methods of regulating expression of GAP-43, thereby regulating axonal growth, and to methods of producing GAP-43 in prokaryotic or eukaryotic hosts cells or organisms. More particularly, the invention is related to a novel membrane- targeting peptide derived from GAP-43, which is capable of regulating membrane binding and growth cone enrichment of GAP-43, and is also capable of directing any desired protein or polypeptide to the membrane of neuronal or non-neuronal cells. The invention further relates to the discovery that GAP-43 and biologically active peptides derived therefrom function as Intemal Regulatory Proteins (IRPs), which can act intracellularly to modulate cell function. The present invention also is related to the clinical in vivo and in vitro diagnostic and therapeutic applications of GAP-43 and its regulatory and membrane-targeting elements in, inter alia, neurological indications in animals including humans.
  • IRPs Intemal Regulatory Proteins
  • GAP-43 is one of the proteins that specifically characterizes growing axons (Skene, Cell 37:697 (1984); Meiri, PNAS USA 83:3537 (1986)).
  • Axonally transported proteins are a small subset of total cellular proteins, and only a few of these vary such that their levels may be envisioned as directly mediating axonal growth (Skene et al., J. Cell. Biol. 89:86 (1981); Benow z et al., J. Neuwscience 3:2153 (1983); Skene, Cell 37:697 (1984); Meiri,
  • GAP-43 is particularly attractive as a candidate since it is primarily a growth cone constituent, where it is bound to the internal surface of growth cone membrane and serves as a substrate for protein kinase C.
  • its level of gene expression correlates well with axonal growth, both in cell culture and in vivo (Basi, Cell 49:185 (1987); Karns, Science 236:597 (1987); Neve, Molec. Brain Res. 2: 177 (1987)).
  • GAP-43 increases to high levels after injury in neurons capable of regeneration, such as toad or goldfish optic nerve or mammalian peripheral nerve, but not after similar injury to mammalian CNS neurons (Skene et al., J. Cell. Biol. 89:96 (1981)).
  • the present inventors have examined the role of GAP-43 in human 20 CNS function and disease.
  • Human GAP-43 cDNA has been cloned, and its developmental and adult distribution examined by assay of post-mortem tissue.
  • the present inventors have discovered that GAP-43 expression persists in discrete regions of the adult, and unexpectedly, that acute ischemic injury is associated with heightened ,25 expression of GAP-43 even in areas where it is normally low.
  • cDNA for rat GAP-43 has been used as a probe to identify and clone cDNA for human GAP-43 from human brainstem and cerebellum libraries.
  • the amino acid sequence for human GAP-43 also has been determined.
  • substantially pure mammalian GAP-43 protein or a functional derivative thereof.
  • rat and human GAP-43 proteins in substantially pure form, as well as the functional derivatives of these proteins.
  • Specific embodiments of the invention comprise substantially pure rat and human GAP-43 proteins and polypeptides having amino acid sequences corresponding to those shown in Figures 2 and 5A, respectively, and their functional derivatives.
  • cDNA comprising a nucleotide sequence as shown in or substantially similar to that shown in Figures 2 or 5A, or functional derivatives thereof.
  • the cDNA of the invention may be incorporated into a suitable expression vector, such as a plasmid, and the vector may be used to transfect a prokaryotic or eukaryotic host cell, which may then express the cDNA under appropriate in vivo, in vitro or in situ conditions, all of which, together with the GAP-43 protein or polypeptide produced thereby, form additional embodiments of the invention.
  • yet another embodiment of the invention provides for a method of producing mammalian GAP-43 protein or polypeptide or a functional derivative thereof, comprising transfecting a prokaryotic or eukaryotic host cell with a vector comprising cDNA encoding mammalian GAP-43 protein or polypeptide, culturing said host cell in a suitable medium and under conditions permitting expression of said mammalian GAP-43 protein or polypeptide, and separating said mammalian GAP-43 protein or polypeptide, or their functional derivatives, from said medium.
  • the substantially pure GAP-43 antigens of the invention the inventors have succeeded in generating antibodies against GAP-43, and such antibodies and their functional and chemical derivatives comprise additional embodiments of the present invention.
  • the GAP-43 antibodies of the invention may be polyclonal or, preferably, monoclonal antibodies, and are suitable for a variety of preparative, diagnostic and therapeutic uses, which are to be understood as forming yet additional invention embodiments.
  • the GAP-43 antigens and antibodies of the invention are well suited for appropriate labeling as, for example, with detectable or therapeutic labels, and for use with other active agents in compositions which may or may not be pharmaceutically acceptable, all of which may be determined as the particular preparative, diagnostic or therapeutic application may require.
  • Such labeled GAP-43 antigens, antibodies and their functional and chemical derivatives, as well as such compositions comprise embodiments of the present invention.
  • the GAP-43 antigens and, particularly, antibodies of the invention, together with their functional and chemical derivatives may be employed in various diagnostic methods known to those of skill. Such methods, including but not limited to immunocytochemical and immunometric methods, form additional embodiments of the invention.
  • a method of determining or detecting mammalian GAP-43 antigen or antibody in a sample comprising contacting a sample suspected of containing GAP-43 antigen or antibody with detectably labeled GAP-43 antibody or antigen, respectively, incubating said sample with said antibody or antigen so as to allow the formation of a GAP-43 antigen-antibody complex, separating the complex thus formed from uncomplexed antigen or antibody, and detecting the labeled complexed antibody or antigen.
  • this embodiment of the invention, and others may be carried out in vivo, in vitro or in situ, as may be desired.
  • kits When used in the preparative, diagnostic or therapeutic methods of the invention, the compounds and compositions of the invention may conveniently be included in a kit, and such kits form yet another embodiment of the present invention.
  • a kit useful for the preparation, purification, isolation, determination or detection of GAP-43 antigen or antibody, or for therapeutic treatment with GAP-43 antigen or antibody comprising carrier means being compartmentalized to receive in close confinement therein one or more container means, wherein one or more of said container means comprises preparatively , detectably or therapeutically labeled GAP-43 antigen or antibody, or their functional or chemical derivatives.
  • the present inventors also have evaluated GAP-43 expression in normal, as well as in damaged or diseased CNS tissue. It has been discovered that in vivo GAP-43 expression varies during development in neural tissue, and that regional variations in GAP-43 expression exist. Further, it has been discovered that GAP-43 expression undergoes significant changes as a result of damage to neural tissue.
  • GAP-43 expression may be enhanced or, if desired, inhibited.
  • GAP-43 expression is enhanced by nerve growth factor, and that this is inhibited by certain steroids.
  • the ability to modulate GAP-43 expression may be of great therapeutic utility in treating mammals, and particularly humans, suffering from damage to, or from disease or dysfunction of, the central or peripheral nervous system, the significance of these discoveries will be readily apparent.
  • the inventors by introducing into non-neural cells cDNA encoding GAP-43 or its functional derivative, the inventors have made the surprising discovery that even non- neural cells can form growth cone-like processes. Again, the potential therapeutic value of this discovery is profound.
  • the present invention comprises methods for evaluating or determining GAP-43 activity and expression in diseased or damaged CNS tissue, as well as in normal CNS tissue.
  • the present invention further comprises methods of treating mammals, including humans, suffering from damaged, diseased or dysfunctioning central or peripheral nervous tissue, and methods of modulating structural remodeling in normal CNS tissue in mammals including humans.
  • the invention comprises a method of inducing expression of GAP-43 in cells, comprising exposing said cells in vivo, in vitro or in situ to an effective amount of nerve growth factor.
  • GAP-43 expression may be induced or enhanced by introducing into non-neural or neural cells cDNA encoding GAP- 43. This may be accomplished in vivo, in vitro or in situ by a variety of means, including transfection, transduction and direct microinjection, all of which form intended non-limiting embodiments of the invention.
  • the cDNA of the invention may be introduced by means of a retroviral or viral vector, or may be attached to any number of cell surface receptor ligands and conveyed with such ligands into the cell. All of these methods, as well as the compositions and vectors comprising GAP-43 cDNA and its functional and chemical derivatives, form additional embodiments of the present invention.
  • yet additional embodiments of the present invention comprise methods of modulating structural remodeling, methods of modulating synaptic plasticity, and methods of modulating the microenvironment of cells, including neuronal and non-neuronal cells, comprising exposing said cells to an effective amount of one or more substances selected from the group consisting of nerve growth factor, steroid and their functional derivatives.
  • GAP-43 surprisingly contains a ten amino acid amino-terminus exon, and that this peptide is responsible for directing GAP-43 to the cell membrane, and especially to the growth cone regions of neuronal cells. It has further been discovered that this ten amino acid membrane-targeting peptide, and its functional derivatives, are capable of directing a desired protein or peptide to the cell membrane, when attached at or near the amino-terminus of such protein or peptide. This surprising discovery applies to proteins and peptides which are normally cytosolic, and not normally membrane-associated.
  • an additional embodiment of the present invention comprises a membrane-targeting peptide, or a functional derivative thereof, capable of directing any desired protein or peptide to the cell membrane of neuronal ⁇ r non-neuronal cells.
  • the membrane-targeting peptide of the invention, or the desired protein or peptide to which it is attached, may be diagnostically or therapeutically labeled.
  • Methods of diagnostic or therapeutic in vivo, in vitro or in situ treatment of neuronal or non-neuronal cells of animals, including humans, using the membrane-targeting peptide form additional embodiments of the present invention.
  • the present invention provides for genomic GAP-43, which has been isolated, and its intron-exon boundaries and transcriptional start sites mapped. Further, it has surprisingly been discovered that the GAP-43 promoter is quite unusual in its structure, and may be useful for the expression of other structural genes.
  • the present invention is directed to the surprising discovery that GAP-43 acts intracellularly to modify the binding capacity of other cell proteins, including that of G 0 .
  • the present invention thus provides for an important new class of internal regulatory proteins ("IRP"), of which GAP-43 is representative, comparable in effect and utility to external cell receptors.
  • IRP internal regulatory proteins
  • synthetic peptides comprising the amino terminus amino acids of GAP-43 also exhibit biological activity as internal regulatory proteins, and compositions comprising such proteins and their use constitute additional embodiments of the invention.
  • the invention is directed to the discovery that these IRP may be used to modulate structural remodeling in a neural cell.
  • the modulation of structural remodeling may result in either inhibition or stimulation of neural growth depending upon the particular
  • the invention further relates to the discovery that modified GP-43, peptides in which cys 3 and cys 4 have been palmitoylated or substituted with amino acids such as THR, ASP or GLU, also modulate the binding of G proteins. These peptides may also be used to modulate structural remodeling in a neural cell.
  • the invention relates to a method for augmenting the activation of a desired protein by, a receptor by administering an IRP.
  • the protein is a G protein.
  • the activation of the G protein occurs in a neural cell.
  • FIG. 1 Hybrid-selected translation of GAP-43 cDNA.
  • the EcoRI insert, GAP43-2 was used to select mRNA by the procedure of Ricciardi et al., PNAS 76:4927 (1979).
  • 0.5 mg of the GAP43-2 insert, or equivalent amounts of nonspecific DNA, the bacterial plasmid pSP65 were spotted onto nitrocellulose and hybridized with 17.5 mg of newborn rat brain polyadenylated [poly(A) + ] RNA in a solution with 65% formamide, 400 mM NaCl, 10 mM 1,4-piperazine diethanesulfonic acid (Pipes) pH 6.4 at 42°C for 16 hours. After being washed in standard saline citrate (SSC) (XI), 0.5%
  • FIG. 1 Nucleotide sequence and predicted amino acid sequence of GAP-43.
  • the cDNA library was generated with RNA from dorsal root ganglia from embryonic day 17-18 rats. Total cellular RNA was isolated by the method of Chirgwin et al, Biochemistry 18:5294 (1979), and poly(A) + RNA was selected with oligo-dT cellulose. Double-stranded cDNA was generated by the ribonuclease H method described by Gubler et al. , Gene 25:263 (1983), ligated to EcoRI linkers, and ligated into the EcoRI site of the lambda phage cloning vectors, lgtlO and lgtll.
  • IPTG isopropyl b-D- thiogalactopyranoside
  • the insert, GAP43-2 was sequenced by using the series of overlapping restriction fragments shown below the sequence by the dideoxynucleotide chain- termination method (Sanger et al., PNAS 74:5463 (1977)).
  • the 3' end of this fragment is the EcoRI site common to the three independent lgtll isolates, which is thought to be an EcoRI site that occurs naturally in the GAP-43 gene. Since none of the clones contained an insert with a polyadenylation sequence, it is likely the EcoRI sites within the cDNA were unsuccessfully methylated during the library construction.
  • the predicted protein sequence for GAP-43 is shown above the DNA sequence.
  • the first methionine in italics was chosen as the start of the coding region for the reasons described hereinafter. It is unlikely that the only other methionine, shown here as amino acid 5, could alternatively serve as the initiation codon.
  • the amino acid residues that were identified by direct protein sequencing from the arginine (R) at amino acid 7 to the isoleucine (I) at amino acid 20 are overlined. The first cycle of sequencing at which the amino acid could be determined with certainty was this arginine. The next amino acid could not be determined with certainty. The inability to sequence the unfragmented protein suggests that the amino terminus may be blocked.
  • RNA (10 mg per sample) was denatured and ran on a 1.2% agarose-formaldehyde gel, transferred to a GeneScreen nylon filter, bound to the filter by ultraviolet cross-linking, and probed with 32 P-labeled GAP43-2 (B).
  • the final wash was SSC (x ⁇ .2), 0.1% SDS at 65 °C.
  • the additives included were (i) none, (ii) 50 ng of NGF per milliliter, (iii) 10 ⁇ 3 M dibutyryl cAMP, and (iv) 50 ng of NGF per milliliter and
  • RNA from newborn brain run as a positive control for the blotting and hybridization procedure.
  • FIG. 4 Developmental regulation and tissue specificity of GAP-43 gene expression.
  • Total cellular RNA was isolated from the designated rat tissues by a modification of the procedure of Chirgwin et al., Biochemistry
  • RNA (10 mg) was denatured, underwent electro ⁇ phoresis in a 1.2% agarose-formaldehyde gel, and was transferred to nitro ⁇ cellulose.
  • the filter was hybridized overmght at 42°C with the EcoRI insert from lgtl 1 GAP43-2 labeled with deoxycytidine 5'-[a- 32 P]triphosphate by nick translation.
  • the final wash was done in SSC (x ⁇ .2), 0.1% SDS at 65° €.
  • RNA samples (i) embryonic day 13 (E13) heart (H), (ii) E13 liver (L), (iii) E13 brain (B), (iv) E13 dorsal root ganglion (DRG), (v) to (viii) embryonic day 17 heart, liver, brain, and dorsal root ganglion; (ix) to (xii) newborn heart, liver, brain, and dorsal root ganglion; (xiii) to (xvi) adult heart, liver, brain and dorsal root ganglion.
  • RNA are shown at the right. Below is hybridization of the same filter with a cDN A probe encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Piechacyk et al., Cell 42:589 (1985)).
  • Figure 5. Nucleotide sequence and deduced amino acid sequence of human GAP-43 cDNA. E: EcoRI; H: Haelll; M: Mspl. The coding region is denoted by thick bar. The scale is 100 bp. Arrows show the overlapping restriction fragments that were sequenced.
  • B Alignment of human, rat GAP-43 and mouse P-57 amino acid sequences. Vertical bars indicate identity, and colons show conservative substitutions.
  • the amino acids are represented by IUPAC-IUB CBN one-letter symbols.
  • the rat sequence is that of Figure 2, and the mouse sequence is from Cimler et al., J. Biol. Chem. 262: 12158 (1987).
  • C Stem-loop structures in the 3'-untranslated region pre- dieted by fold program of Zuker et al. , Nucl. Acids Res. 9: 133 (1981).
  • FIG. 1 Northern blot showing the regional restriction of GAP-43 expression with maturation. Ten mg total RNA from 8-day-old, 16-year-old, and 64-year-old brain regions were loaded in each lane and the blot was probed with human GAP-43 probe Cla as described in Example II. The positions of 18S and 28S rRNA bands are indicated.
  • FIG. 7 Northern blot showing that GAP-43 expression increases in the wake of an ischemic event.
  • A Ten mg of RNA from different brain regions of a patient with a stroke in Area 17 (visual cortex). Expression in A17 has increased to levels comparable to the highest in the brain (Al 1).
  • B Ten mg of RNA from Area 3, 1 ,2,5 from three patients, all ran and blotted on the same blot with an unrelated band excised between lanes 2 and 3. Lanes 1 and 2 were histologically normal, whereas 3 included a small stroke, and shows an increase in GAP-43 expression.
  • Al Higher magnification of infarcted region in B showing diffuse infiltration of tissue by lipid-laden macrophages and reactive astrocytes. There are no remaining neurons in this region (xl60).
  • A2 Normal adjacent cortex with abundant histopathologically intact neurons (x300).
  • B Lower magnification view of the visual cortex with an organizing ischemic infarct (10 to 14 days old) involving one gyrus (arrowheads) and intact cortex in the adjacent gyrus (arrows) (xl2).
  • C In normal visual cortex GAP-43 expression is restricted to a few scattered neurons by darkfield examination (arrowheads).
  • Figure 9 Enhanced GAP-43 expression in neurons of cerebellar cortex several days following a bout of severe hypotension and hypoxia. All sections were processed simultaneously.
  • A Normal adult cerebellar cortex showing absence of detectable GAP-43 labeling.
  • B Post-ischemic cerebellar cortex showing markedly increased expression of GAP-43 in the Puri ⁇ nje cell and outer granule cell layers.
  • C A section adjacent to B hybridized with the sense strand probe as control (all x315).
  • Figure 10 Effect of GAP-43 on process formation in CHO cell lines. Empty bars represent cells with processes in 4 CDM8-transfected lines and solid bars represent 4 cell lines expressing GAP-43. Cell lines were obtained as described in Example V. The percentage of cells with processes was assayed by plating CHO cells onto poly-D-lysine-coated coverslips. Cells with processes longer than 20 microns were scored as positive. To ensure comparability, all assays were performed within the time window that extended from 30 to 45 minutes after plating. An important component of this assay was the time window selected, since, after longer plating times, or as cells reached confluence, processes were much less evident. As many cells as possible were counted during this time window, and all cells examined were included.
  • the number of cells counted for the different lines was: IA, 406; 1B, 408; 2A, 287; 2B, 303; 5E, 234; 4, 333; 12, 156; 14, 161.
  • the proportion of cells with processes in GAP-43 expressing cell lines was significantly greater than in controls (p ⁇ 0.001).
  • FIG. 11 Schematic representation of experiments demonstrating that the amino-terminus exon is responsible for directing the GAP-43 protein to the cell membrane, and that it directs membrane targeting of chloramphenicol acetyl transferase.
  • the left column (“CONSTRUCTION”) indicates the gene construction used for transfection of COS, NIH 3T3, CHO or PC12 cells.
  • the right column (“MEMBRANE”) indicates whether the expressed protein or fragment was membrane-associated (+) or not (-), as assayed by sub- cellular fractionation followed by Western blotting, direct immunofluores ⁇ cence, or both.
  • GAP GAP-43 gene
  • GAP(-intern.) GAP constructions lacking substantial portions of exon 2
  • GAPtag carboxy-terminus region of the GAP-43 gene
  • the nucleotides encoding the first four amino acids of GAP-43 were deleted (GAP(- 1-4))
  • the expressed protein fragment was not membrane- associated.
  • CON had no GAP- 43
  • Brain membranes (BR) had GAP-43 of the same molecular weight as that from the transfected gene.
  • A A linear depiction of the GAP-43 gene in the 5' to 3' orientation. Representations of the phage inserts that were used for mapping are shown. The three exons are depicted as vertical bars. The sites shown are for restriction endonucleases BamHI (B), Kpnl (K), and Sad (S).
  • FIG. 14 Sequence of the GAP-43 promoter region. Nucleotide position + 1 denotes the A of the initiating ATG codon of the GAP-43 protein.
  • This sequence includes the variably sized first exon which ends at +30.
  • Major transcriptional start sites are denoted by arrows. Purine residues have been underscored by asterisks.
  • the consensus Pit-1 binding site is overlined.
  • GAP-43 promoter region from -518 to +85 showing locations of restriction sites and the major homopurine-homopyrimidine regions (thickened and labeled I, II, and III).
  • FIG. 1 DRG Growth Cones. DRG growth cones on laminin- coated glass after 30 min. incubation (a) with F12 medium only, (b) with 0.3 mg/ml of chick E10 brain-derived crude collapsing activity, and (c) with the same concentration of collapsing activity as in after PTX-pretreatment for 2 hours.
  • FIG. 1 DRG Growth Cone Collapse Induced By Brain Membrane Extracts And Mastoparan Is Blocked By PTX.
  • Chick E7 DRG explants were treated with no PTX (closed square), 100 ng/ml PTX (closed circle), or 200 ng/ml PTX (closed triangle), and then CHAPS-solubilized embryonic brain extracts were added. The percentage of collapsed growth cones -is shown as a function of protein concentration in the extract added,
  • FIG. 19 DRG Growth Cone Collapse Induced by Myelin Is Blocked By Pertussis Toxin.
  • the percentage of collapsed growth cones of E7 DRG was scored in the absence (left pair) and the presence (right pair) of 100 ng/ml PTX.
  • the collapse assay was performed as in Figure 21(a), except that the incubation time with crude collapsing activity is 1 hour instead of 30 min. (hatched bar). In the control, F12 medium only was added to DRG explants (open bar). Data are shown as the average and S.E.M. of three to six independent assays. Pertussis toxin itself does not affect the percentage of collapsed growth cones.
  • FIG 20 Pertussis Toxin Partially Blocks Retinal Growth Cone Collapse Induced By Brain Membrane Extracts.
  • the effects of CHAPS- solubilized membranes from chick embryonic brain are shown on retinal growth cones in the control (left pair) and after PTX-pretreatment (right pair).
  • the temporal half of chick E7 retinae were isolated and cut into small fragments. Each fragment was explanted onto laminin-coated plastic chamber slides.
  • the collapse assay using brain membrane extracts was performed as in Figure 18a (hatched bar). In the control, only F12 medium was added to the retinal explants (open bar).
  • retinal explants were incubated with F12 medium containing PTX (100 ng ml) for 2 hour prior to the addition of the membrane extracts. Data are shown as the average and S.E.M. of three to six independent assays.
  • FIG. 21 Modulation of (35S)GTP7S binding to G 0 by GAP-43 and GAP-43 peptides.
  • the same concentration of GAP-43 without G 0 binds no detectable (35S)GTP ⁇ S.
  • Variation of the GAP-43 concentration demonstrates an EC50 of 150 nM for this effect (A, right).
  • the range of EC50 was 150-800 nM, perhaps reflecting partial inactivation of GAP-43 during purification.
  • GAP(35-53) ATKIQASFRGHITRKKLKD
  • GAP(53-69) DEKKGDAPAAEAEAKEK
  • GAP(210-226) ARQDEGKEDPEADQEHA
  • the stimulation of binding by the 1-24 peptide is saturable with an EC50 of 20 uM (B, right).
  • a peptide with threonine in place of cysteine at positions 3 and 4 has no effect on (35S)GTP7S binding to Go.
  • the IC50 for the 1-10 peptide is 20 uM (C, right).
  • Panel D shows (35S)GTP7S binding to G 0 as a function of GAP-43 concentration in the presence or absence of 50 uM 1-10 peptide. Note that GAP-43 reverses inhibition by the peptide, and that the EC50 for GAP-43 is shifted from 600 nM to 12 uM by this concentration of peptide. This -is consistent with direct competition between the 1-10 peptide and GAP-43. -
  • Non-specific binding was determined in the presence of 10 uM unlabeled GTP7S and subtracted from total binding to determine specific binding. All assays were performed in duplicate. An average assay tube contained 800,000 cpm and yielded 30,000 cpm total binding and 1500 cpm non-specific binding.
  • the addition of GAP-43 increased specific binding without effect on non-specific binding.
  • GAP-43 activated two different samples of G c and one of alphao, all prepared in Lubrol PX. However, the extent of the activation varied among the preparations. Activation by GAP-43 appears to be sensitive to the detergent used in the purification of G 0 and the composition of the binding buffer. GAP-43 was purified by a modification of the method of Zwiers et al.
  • FIG. 22 Homology between GAP-43 amino terminus and the cytoplasmic tail of G-linked receptors.
  • the first 10 amino acids of GAP-43 were compared to the sequence of the G-linked receptor family.
  • Homology between the first 7 amino acids of G AP-43 and the amino terminal segment of the cytoplasmic tail of a number of G-linked receptors is shown.
  • Residues which conform to the consensus sequence hydrophobic LEU CYS CYS X basic-basic are shaded.
  • the cysteines illustrated in the receptors are located approximately 13 amino acids distal to the last transmembrane region, correspond to cys341 of the B2-adrenergic receptor, and are aligned as described in Higashijima, et al.
  • FIG. 23 Dose-dependent reduction by calmodulin of GAP-43- stimulated GTP7S binding to G 0 .
  • Vertical axis for both panels is GTP7S binding expressed as a percentage of control.
  • C calmodulin
  • G GAP-43.
  • Addition of GAP-43 to G neurons (Lane 2) increased GTP7S binding to over 200% as compared to G c alone (Lane 1).
  • Addition of calmodulin reduced this binding to control levels (Lane 5). No GTP7S binding was seen in the absence of G 0 , using GAP-43 alone (Lane 3) or GAP-43 plus calmodulin (Lane 4).
  • Right panel calmodulin concentration, ⁇ M, demonstrating the dose-dependency of the effect of calmodulin on GTP7S binding to G 0 .
  • FIG 24 Filopodial formation by non-neuronal cells transfected with GAP-43.
  • A Stably transfected CHO cells were examined for filopodial formation after 15 minute incubation on poly-L-lysine coated glass. Two control cells from lines expressing CDM8 (a) or CAT (b) exhibit no filopodia, but several GAP-43-expressing cells (c-e) do form filopodia under the same conditions. The percentage of CHO cells with filopodia in each population is quantitated in Figure 30; the cells shown here are representative of positive and negative cells. The scale bar is 8 ⁇ .
  • B COS cells expressing (CAT ( ⁇ ) or GAP-43 (A) were analyzed for filopodial formation as in Experimental Procedures. Note that at the shortest times examined, more GAP-43 cells have filopodia, and that few cells have filopodia after 30 minutes. The zero time point represents cells that were fixed before plating. This is one of four experiments with similar results.
  • Figure 25 Decreased spreading of non-neuronal cells transfected with GAP-43.
  • A COS cells expressing CAT ( ⁇ ) or GAP-43 (•) were plated for the indicated times on poly-L-lysine coated glass, and then analyzed for the percentage of cells spread. Note that more spread cells are detected in the control CAT transfection. This is one of four experiments with similar results.
  • B Four control A431 cell lines (a,b,c,d) exhibit a more flattened, spread phenotype as compared to four GAP-43-expressing A431 cell lines (e,f,g,h). The cells were fixed 2 hours after plating. The scale bar is 50 ⁇ .
  • C Control or GAP-43-expressing A431 cell lines were assayed for cell spreading or laminin-coated glass. The data are averaged from 6 control and
  • FIG. 26 The first 10 amino acids of GAP-43 are necessary and sufficient to induce cell shape changes.
  • COS cells expressing the indicated proteins were analyzed for filopodia (A) and spreading (B).
  • A filopodia
  • B spreading
  • GAP- 43, GAP-43 with a deletion from 40-189, GAP-43(l-40)/CAT fusion and GAP-43(1-10)/CAT are active in both assays, but that GAP-43 with a deletion form 2-5 and GAP-43(l-6)/CAT are indistinguishable from the CAT control.
  • the values shown are means ⁇ SEM for 3-7 separate transfections.
  • the COS cell filopodia results for CAT, GAP-43, GAP-43 with a deletion from 40-189, and the GAP-43(l-40)/CAT were confirmed in stably transfected CHO clonal cell lines.
  • the control data includes 3 cell lines transfected with the neo gene alone and three lines expressing CAT as well.
  • FIG. 27 Basic residues at position 6 and 9 are required for GAP-43 modulation of cell shape.
  • COS cells expressing the indicated CAT, GAP-43 or GAP-43(1-10)/CAT proteins were analyzed for filopodia (A) and for spreading (B). Note that GAP-43, GAP-43 with arg 7 changed to gly, GAP-
  • FIG. 29 A GAP-43/CAT fusion protein without cell shape- modulating activity is localized in membrane fractions.
  • COS cells expressing CAT (a), GAP-43(l-40)/CAT (b), (1-10)/CAT (c) or GAP-43(l-6)/CAT (d) were separated into membrane (top) and soluble (bottom) fractions and analyzed for CAT immunoreactivity on blots of SDS-PAGE gels. Membrane and soluble fractions from 10 6 cells were loaded in each lane. Note that all three fusion proteins are detected in the membrane fraction but CAT is in the cytosolic fraction.
  • FIG. 30 Basic residues at position 6, 7 and 9 of GAP-43 are not required for membrane binding.
  • COS cells expressing GAP-43 (a), GAP-43 with arg 6 changed to gly (b), GAP-43 with arg 7 changed to gly (c), or GAP-43 with lys 9 changed to gly (d) were separated into membrane (m) and cytosolic (c) fractions and analyzed by immunoblot for GAP-43 immunoreactivity. Note that all four proteins are present primarily in the membrane fraction. Material from 10 6 cells was loaded in each lane.
  • FIG. 31 The amino terminus of GAP-43: functional domains and comparison to the G protein activator region of receptor proteins.
  • the first ten amino acids of GAP-43 contain a membrane targeting signal (residues 1-6,
  • FIG 33 and a G protein activator region (residues 1-10).
  • the G protein activator region is compared to that of the insulin-like growth factor II receptor (Okamoto et l., Cell 62:709-717 (1990)), and the /32-adrenergic receptor (Okamoto et al. , Cell 67:723-730 (1991a)).
  • the arrangement of basic residues identified for G protein activation identified by Nishimoto and colleagues (Okamoto et al., Cell 62:709-717 (1990); Okamoto et al, Cell 67:723-730 (1991a)) is shown at the bottom.
  • the second basic in the BBxB, or BBxxB motif is boxed but not shaded because an IGF-II peptide with this residue altered is still active (Okamoto et al. _ Biochem. Biophys. Res. Comm. 779: 10-16 (1991b)).
  • GAP-43 with arg 7 changed to gly is still active in altering cell shape ( Figure 31).
  • the initiator methionine of GAP-43 is boxed to indicate the charge of the amino group at that position.
  • FIG. 32 Filopodial Formation in COS Cells Transiently Transfected With Expression Vectors Encoding CAT, GAP-43, or GAP-CAT
  • Lanes (a)-(d) are membrane fractions. Lanes (e)-(h) are from soluble fractions. Lanes (a) and (e) are from the same CAT transfection; (b) and (f) from GAP (1-40)-
  • FIG. 34 GAP-43 Peptides (100 ⁇ M) Stimulate GTP7S Binding to G 0 .
  • FIG. 36 Neurite Outgrowth From Control and Briefly Permeabilized N1E-115 Cells in the Presence of the Indicated GAP-43 Peptides.
  • FIG. 37 GAP-43 and Receptor Stimulate G Q Synergistically - (A) The purity of the rat brain GAP-43 preparation used in all experiments is demonstrated by Coomassie Blue staining of an SDS-PAGE gel. The mobility of M r standards of 97, 66, 45, 31, 21.5, and 14 kDa is indicated at right.
  • CCh carbachol
  • FIG 40 GAP-43 Does Not Block IP 3 -Induced Desensitization. A continuous current trace from one oocyte injected with the high concentration of GAP-43 thirty minutes previously is shown. 5HT produces a large response which returns to baseline after 9 minutes, and this is followed by a current response to the intracellular injection of 4 pmol IP 3 . Subsequent bath application of 5HT, or intracellular injection of IP 3 in this GAP-43- injected oocyte produces no change in current. Similar results were observed in 3 of 3 cells examined in this protocol. Figure 41. GAP-43 inhibits forskolin-stimulated cAMP levels in
  • A431 cells were stably transfected with a neo expression cassette alone (control), or with a GAP-43 anda neo expression vector (GAP-43- expressing cells). Cyclic AMP levels were measured in the presence of IBMX and the indicated agents. The data are from 6 control lines and 4 GAP-43 lines.
  • FIG 42 GAP-43 inhibits isopreternol-stimulated cAMP levels in A431 cells. Control and GAP-43-expressing cell lines as in Figure 41 were assayed for cAMP levels in the presence of the indicated agents.
  • cloning is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule.
  • it is necessary to employ methods for generating DNA fragments, for joining the fragments to vector molecules, for introducing the composite DNA molecule into a host cell in which it can replicate, and for selecting the clone having the target gene from amongst the recipient host cells.
  • cDNA is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a cDNA clone means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire genome of an organism. Such a cDNA library may be prepared by methods known to those of skill, and described, for example, in Maniatis et al., Molecular
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene.
  • Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines.
  • vector is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • DNA expression vector is meant any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome.
  • DNA expression vectors include bacterial plasmids and phages. Preferred for the purposes of the present invention is the lambda gtll expression vector.
  • substantially pure is meant any antigen of the present invention, or any gene encoding any such antigen, which is essentially free of other antigens or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule, such as any of the cDNA sequences of the present invention is meant to refer to any nucleotide subset of the molecule.
  • a “variant” of such molecule is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a molecule is said to be "substantially similar" to another molecule if the sequence of amino acids in both molecules is substantially the same.
  • Substantially similar amino acid molecules will possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule.. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc.
  • a “functional derivative" of a gene of any of the antigens of the present invention is meant to include “fragments,” “variants,” or
  • analogues of the gene which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity.
  • a DNA sequence encoding GAP-43 or its functional derivatives, or the membrane-targeting peptide or functional derivatives thereof, may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and ligation with appropriate ligases. Techniques for such manipulations are disclosed by Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, NY (1982) and are well known in the art.
  • a nucleic acid molecule such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are “operably linked” to nucleotide sequences which encode the polypeptide.
  • An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene expression.
  • regulatory regions needed for gene expression may vary from organism to organism, but shall in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal the initiation of protein synthesis.
  • Such regions will normally include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like.
  • the non-coding region 3' to the gene sequence coding for the protein may be obtained by the above-described methods.
  • This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation.
  • the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of the GAP-43 gene sequence, or (3) interfere with the ability of the GAP-43 gene sequence to be transcribed by the promoter region sequence.
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.
  • the present invention encompasses the expression of the GAP-43 protein (or a functional derivative thereof) in either prokaryotic or eukaryotic cells.
  • Preferred prokaryotic hosts include bacteria such as E. coli, Badllus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.
  • the most preferred prokaryotic host is E. coli.
  • Other enterobacterium such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may also be utilized. Under such conditions, the GAP-43 will not be glycosylated.
  • the procaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • GAP-43 protein or a functional derivative thereof in a prokaryotic cell (such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.)
  • a prokaryotic promoter such as, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.
  • Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage 1, the bla promoter of the b-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325, etc.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage 1 (P L and P R ), the trp, recA, lacZ, loci, and gal promoters of E. coli, the a-amylase (Ulmanen, et al., J. Bacteriol. 162:176-182 (1985)) and the s-28-specific promoters of B. subtilis (Gilman, et al., Gene 32:11-20
  • ribosome binding sites are disclosed, for example, by Gold, et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).
  • Most preferred hosts are eukaryotic hosts including yeast, insects, fungi, mammalian cells (especially human cells) either in vivo, or in tissue culture. Mammalian cells provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Mammalian cells which may be useful as hosts include cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/O-AG14 or the myeloma P3x63Sg8, and their derivatives. Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332, that may provide better capacities for correct post-translational processing. COS cells also are convenient eukaryotic hosts for GAP-43 expression, as well as for study of the regulation of GAP-43 expression, and are preferred for this purpose.
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, etc., may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression of the genes can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical regulation, e.g., metabolite.
  • Yeast provides substantial advantages in that it can also carry out post- translational peptide modifications including glycosylation.
  • Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • Any of a series of yeast gene expression systems incorporating promoter and termination elements from the actively expressed genes coding for glycolytic enzymes produced in large quantities when yeast are grown in mediums rich in glucose can be utilized.
  • Known glycolytic genes can also provide very efficient transcriptional control signals.
  • the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.
  • GAP-43 or functional derivatives thereof in insects can be achieved, for example, by infecting the insect host with a baculovirus engineered to express GAP-43 by methods known to those of skill.
  • sequences encoding GAP-43 may be operably linked to the reguiatory regions of the viral polyhedron protein (Jasny, Sdence 238: 1653 (1987)).
  • Infected with the recombinant baculovirus, cultured insect cells, or the live insects themselves, can produce the GAP-43 protein in amounts as great as 20 to 50% of total protein production.
  • caterpillars are presently preferred hosts for large scale GAP-43 production according to the invention.
  • eukaryotic regulatory regions Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer, etal., J. Mol. Appl. Gen. 7:273-288 (1982)); the TK promoter of Herpes virus (McKnight, Cell 37:355-365 (1982)); the SV40 early promoter (Benoist, etal., Nature (London) 290:304-310 (1981)); the yeast ga!4 gene promoter (Johnston, et al., Proc. Natl. Acad. Sd. (USA) 79:6971-6975 (1982); Silver, et al., Proc. Natl. Acad. Sd. (USA) 87:5951-
  • the GAP-43 encoding sequence and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic cell either as a non- replicating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous replication, the expression of the GAP- 43 protein may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integra ⁇ tion of the introduced sequence into the host chromosome.
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for prototropy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or intro ⁇ quizd into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include those described by Okayama, Mol. Cel. Biol. 3:280 (1983).
  • the introduced sequence will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this purpose. Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, xVX.
  • Such plasmids are, for example, disclosed by Maniatis, et al. (In: Molecular Cloning, A .Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • Badllus plasmids include pC194, pC221, pT127, etc.
  • Such plasmids are disclosed by Gryczan, (In: The Molecular Biology of the Badlli, Academic Press, NY (1982), pp. 307-329).
  • Suitable Streptomyces plasmids include pUlOl
  • Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, et al., Miami Wntr. Symp. 79:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyce: Life Cycle and Inheritance,
  • the vector or DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, including such biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran, and such mechanical means as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al., Sdence 240(4858: 1538 (1988)), etc.
  • biochemical means as transformation, transfection, conjugation, protoplast fusion, calcium phosphate-precipitation, and application with polycations such as diethylaminoethyl (DEAE) dextran
  • mechanical means such as electroporation, direct microinjection, and microprojectile (biolistic) bombardment (Johnston et al., Sdence 240(4858: 1538 (1988)), etc.
  • recipient cells After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression of the cloned gene sequence(s) results in the production of the GAP-43 protein, or in the production of a fragment of this protein. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
  • the expressed protein may be isolated and purified in accordance with conventional conditions, such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.
  • the invention also relates to cloned genes which encode a fusion protein comprising GAP-43 or fragment thereof and a detectable enzyme such as beta-galactosidase, or any desired homologous or heterologous protein or peptide.
  • a detectable enzyme such as beta-galactosidase
  • Methods for producing such fusion proteins are taught, for example, Bai, etal., J. Biol. Chem. 267:12395-12399 (1986), or Huynh, et al., "Con ⁇ struction and Screening cDNA Libraries in lgtlO and lgtll," in DNA Cloning Techniques: A Practical Approach, D. Glover (ed.), IRL Press, Oxford,
  • the GAP-43, functional derivative thereof, or fusion protein comprising GAP-43 or fragment thereof and a detectable enzyme or desired protein or peptide may be isolated according to conventional methods known to those skilled in the art.
  • the cells may be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by column chromatography, for example, on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • the GAP-43 or functional derivative thereof, or fusion protein comprising GAP-43 and a detectable enzyme or desired protein or peptide may be isolated by the use of anti-GAP-43 antibodies, or by the use of antibodies directed against the detectable enzyme or desired protein or peptide.
  • anti-GAP-43 antibodies may be obtained by well-known methods, some of which as mentioned hereinafter.
  • the preparation of polyclonal rabbit anti-GAP-43 sera is disclosed in the examples portion of the present specification.
  • Another embodiment of the present invention comprises antibodies against the GAP-43 protein.
  • antibody (Ab) or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
  • the antibodies of the present invention may be prepared by any of a variety of methods.
  • cells expressing the GAP-43 protein, or a functional derivative thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies that are capable of binding GAP-43.
  • the antibodies of the present invention are monoclonal antibodies.
  • Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler etal., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas,
  • Such procedures involve immunizing an animal with GAP-43 antigen.
  • the splenocytes of such animals are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention.
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands, et al. (Gastroenterology 80:225-232 (1981).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the GAP-43 antigen. DEPOS ⁇ OF HYBRIDOMA CELL LINE
  • the preferred monoclonal antibodies of this invention are those having the specificity of the monoclonal antibody designated MAb anti-GAP-43 (H5).
  • the invention comprises hybridoma strains which produce the monoclonal antibodies of the invention.
  • the preferred hybridoma cell line according to the invention is designated H-5, which produces monoclonal antibody designated MAb anti-GAP-43 (H5).
  • the H5 cell line has been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, USA 20851 on 21 December 1989, and given accession number ATCC HB 10316.
  • the antibodies of the present invention are well suited for use in standard immunodiagnostic assays known in the art, including such immunometric or "sandwich” assays as the forward sandwich, reverse sandwich, and simultaneous sandwich assays.
  • the antibodies of the present invention may be used in any number of combinations as may be determined by those of skill without undue experimentation to effect immunoassays of acceptable specificity, sensitivity, and accuracy for the GAP-43 antigen or equivalents thereof.
  • detecting it is intended to include determining the presence or absence of a substance or quantifying the amount of a substance.
  • the term thus refers to the use of the materials, compositions, and methods of the present invention for qualitative and quantitative determinations.
  • an anti-idiotypic antibody is an antibody which recognizes unique determinants present on the antibody produced by the clone of interest.
  • the anti-idiotypic antibody is prepared by immunizing an animal of the same strain used as the source of the monoclonal antibody with the monoclonal antibody of interest. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing antibody to these idiotypic determinants (anti-idiotypic antibody).
  • anti-idiotypic antibody By using the anti- idiotypic antibody of the second animal, which is specific for the monoclonal antibodies produced by a single clone, it is then possible to identify other clones used for immunization.
  • Idiotypic identity between the product of two clones demonstrates that the two clones are identical with respect to their recognition of the same epitopic determinants.
  • the anti-idiotypic antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti anti-idiotypic antibody which will be epitopically identical to the original MAb.
  • the monoclonal antibodies of the present invention may be used to induce anti-idiotypic Abs in suitable animals, such as BALB/c mice. Spleen cells from these animals are used to produce anti-idiotypic hybridoma cell lines. Monoclonal anti-idiotypic Abs coupled to KLH are used as "immunogen" to immunize BALB/c mice. Sera from these mice will contain anti anti-idiotypic Abs that have the binding properties of the original Ab specific for the shared epitope. The anti-idiotypic MAbs thus have idiotopes structurally similar to the epitope being evaluated.
  • the hybrid cells may be cultivated both in vitro and in vivo. High in vivo production makes this the presently preferred method of culture. Briefly, cells from the individual hybrid strains are injected intraperitoneally into pristane-primed BALB/c mice to produce ascites fluid containing high concentrations of the desired monoclonal antibodies. Monoclonal antibodies of isotype IgM or IgG may be purified from cultured supematants using column chromatography methods well known to those of skill in the art.
  • the antibodies of the present invention are particularly suited for use in immunoassays wherein they may be utilized in liquid phase or bound to a solid phase carrier.
  • the antibodies in these immunoassays can be detectably labeled in various ways.
  • labels and methods of labeling There are many different labels and methods of labeling known in the art. Examples of the types of labels which can be used in the present invention include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and metal chelates. Those of ordinary skill in the art will know of other suitable labels for binding to antibodies, or will be able to ascertain the same by the use of routine experimentation. Furthermore, the binding of these labels to antibodies can be accomplished using standard techniques commonly known to those of ordinary skill in the art. One of the ways in which antibodies of the present invention can be detectably labeled is by linking the antibody to an enzyme.
  • enzymes which can be used to detectably label the antibodies of the present invention include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotin-avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • the presence of the detectably labeled antibodies of the present invention also can be detected by labeling the antibodies with a radioactive isotope which then can be determined by such means as the use of a gamma counter or a scintillation counter.
  • Isotopes which are particularly useful for the purpose of the present invention are 3 H, 125 I, 32 P, S, 14 C, 51 Cr, Cl, 57 Co, ⁇ Co, ⁇ e and 75 Se.
  • fluorescent labeling compounds fluoroscein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibodies of the invention also can be detectably labeled using fluorescent emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibodies of the present invention also can be detectably labeled by coupling them to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of the chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent antibody is determined by detecting the presence of lumines ⁇ cence.
  • Important bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the assay to be used.
  • kits form are many, and include, for example, competitive and non-competitive assays.
  • Typical examples of assays which can utilize the antibodies of the invention are radioimmunoassays (RIA), enzyme immunoassays (ELA), enzyme-linked immunosoibent assays (ELISA), and immunometric, or sandwich, immuno ⁇ assays.
  • RIA radioimmunoassays
  • ELA enzyme immunoassays
  • ELISA enzyme-linked immunosoibent assays
  • immunometric or sandwich, immuno ⁇ assays.
  • immunometric assay or "sandwich immunoassay,” it is meant to include simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those skilled in the art. Those of skill will also appreciate that the antibodies of the present invention will be useful in other variations and forms of assays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention. ⁇ 15-
  • buffers can be used as "blockers."
  • concentration of the "blockers” (normally 1-100 microgs/microl) is important, in order to maintain the proper sensitivity yet inhibit any unwanted interference by mutually occurring cross reactive proteins in human serum.
  • buffer system containing the "blockers” needs to be optimized.
  • Preferred buffers are those based on weak organic acids, such as imidazole, HEPPS, MOPS, TES, ADA, ACES, HEPES, PIPES, TRIS, and the like, at physio ⁇ logical pH ranges.
  • Somewhat less preferred buffers are inorganic buffers such as phosphate, borate or carbonate.
  • known protease inhibitors should be added (normally at 0.01-10 microgs/ml) to the buffer which contains the
  • solid phase immunoadsqrbents There are many solid phase immunoadsqrbents which have been employed and which can be used in the present invention.
  • Well known immunoadsorbents include glass, polystyrene, polypropylene, dextran, nylon and other materials, in the form of tubes, beads, and microtiter plates formed from or coated with such materials, and the like.
  • the immobilized antibodies can be either covalently or physically bound to the solid phase immunoadsorbent, by techniques such as covalent bonding via an amide or ester linkage, or by adsorption.
  • Those skilled in the art will know many other suitable solid phase immunoadsorbents and methods for immobilizing antibodies thereon, or will be able to ascertain such, using no more than routine experimentation.
  • labels such as radionuclides may be bound to the antibodies of the present invention either directly or by using an intermediary functional group.
  • An intermediary group which is often used to bind radioisotopes which exist as metallic cations to antibodies is diethylenetriaminepentaacetic acid (DTPA).
  • DTPA diethylenetriaminepentaacetic acid
  • Typical examples of metallic cations which are bound in this manner are: "Tc, 123 I, U1 IN, I, "Ru, 67 Cu, 67 Ga and ⁇ Ga.
  • the antibodies of the invention can also be labeled with non- radioactive isotopes for purposes of diagnosis. Elements which are particularly useful in this manner are ⁇ Gd, 55 Mn, 162 Dy, 52 Cr and ⁇ Fe.
  • the antibodies of the present invention also may be used for immunotherapy in animals, including humans, having a disorder, such as a benign or cancerous neoplasia, which expresses the GAP-43 antigen whh epitopes reactive with the antibodies of the present invention.
  • a disorder such as a benign or cancerous neoplasia, which expresses the GAP-43 antigen whh epitopes reactive with the antibodies of the present invention.
  • the antibodies of the present invention may be unlabeled or labeled with a therapeutic agent.
  • therapeutic agents which can be coupled to the antibodies of the invention for immunotherapy are drugs, radioisotopes, lectins and toxins.
  • Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. Ricin is a toxic lectin which has been used immunotherapeutically. This use is accomplished by binding the alpha-peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site-specific delivery of the toxic defect. This is described, for example, in Vitetta et al., Sdence 238: 1098 (1987), and Pastan et al., Adv. Allergy 47: 641 (1986). Toxins are poisonous substances produced by plants, animals or microorganisms that, in sufficient dose, are often lethal.
  • Diphtheria toxin for example, is a protein produced by Corynebacterium diphtheria. This toxin consists of an alpha and a beta subunit which under proper conditions can be separated. The toxic alpha component can be bound to antibody and used for a site-specific delivery.
  • radioisotopes which can be bound to the antibodies of the present invention for use in immunotherapy are: 125 Um, 131 I, ⁇ Y, 67 Cu, 2l7 Bi, 2U At, 212 Pb, 7 Sc and 109 Pd.
  • the expressed GAP-43 antigen normally is confined within the cell membrane. Accordingly, those of skill will recognize that in vivo diagnostic and therapeutic methods employing the antibodies of the invention may require some mechanism by which such antibodies can detect GAP-43 on the intracellular membrane.
  • One such method is to introduce the antibodies or fragments thereof into the cell's membrane or into the cell itself across the cell membrane. This may be accomplished, for example, by attaching the antibody to a ligand for which the target cell contains receptor sites. The antibody can thus be transported into the cell membrane or across the cell membrane along with the ligand.
  • a carrier ligand will depend on several factors, as those of skill will appreciate. These include, for example, the kinetics of the ligand and its receptor, and of overall transport, which may include passive or active, with actively transported ligands preferred.
  • the means of attaching the antibody to the ligand also will vary within limits, and may be, for example, covalent or ionic, bearing in mind that such attachment should not unacceptably alter ligand-receptor affinity.
  • receptors suitable for such applications include the receptor for low density lipoprotein (LDL), which has been shown to contain all the information necessary for receptor endocytosis, Davis et al., J. Cell
  • the ligand may itself be an antibody or fragment specific for the receptor, to which may be conjugated the antibody of the invention.
  • those of skill may find it particularly desirable to employ antibody fragments of the invention (such as, for example, Fab or F(ab') 2 frag ⁇ ments), which are less likely to interfere with the ligand-receptor interaction, and may be more easily transported across the cell membrane.
  • Single-chain antibodies may prove preferable for these and other reasons, as will be appreciated by those of skill.
  • an antibody When an antibody is to be transported into the cell's membrane or into the cell as described above, it will be preferred to diagnostically or thera- Commissionically label the antibody in such a way that the label will be relatively more effective when the antibody is bound to its antigenic site on the GAP-43 protein. This may accomplished, for example, by employing a label which becomes active or detectable as a result of formation of the antigen-antibody complex. Alternatively, the antibody itself may be labeled in such a way that antigen-antibody complex formation induces a conformational change in the antibody to expose or more fully expose the previously unexposed or less fully exposed label. All of the above criteria, and others, will be apparent to those of skill in carrying out these aspects of the invention.
  • liposomes having the antibodies of the present invention in their membrane to specifically deliver the antibodies to the target area.
  • These liposomes can be produced so that they contain, in addition to the antibody, such immunotherapy agents as drugs, radioisotopes, lectins and toxins, which would be released at the target site.
  • the antibodies, and preferably, the GAP-43 encoding nucleotide sequences may be introduced into neural cells for diagnostic or therapeutic purposes is by the use of viral, including retroviral, vectors.
  • viral including retroviral, vectors.
  • suitable viruses may be mentioned the various herpes viruses.
  • Suitable retroviruses include human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Other suitable viruses and retroviruses are well known to those of skill.
  • an attenuated viral or retroviral strain For the purposes of the present invention, it may be preferred to employ an attenuated viral or retroviral strain.
  • retroviruses having attenuated cytopathicity such as HIV-2 ST (Kong et al, Sdence 240(4858): 1525 (1988)) or HI ⁇ (Evans et al., Sdence 240(4858): 1523 (1988)), which enter neural cells by a CD4-dependent mechanism (Funke et al., J. Exp. Med. 165: 1230 (1987)).
  • the neurobiology of HIV infections is described, for example, in Johnson et al, FASEB J.
  • CD4 is known to have a variant transcript in the human brain, with its highest content in forebrain (Maddon et al, Cell 47: 333
  • the DNA sequences which encode GAP-43, or a fragment thereof, may be used as DNA probes to isolate the corresponding antigen in humans according to the above-described methods for isolation of rat GAP-43 with labeled probes.
  • the human antigen genes may then be cloned and expressed in a host to give the human antigen. This human antigen may then be used in diagnostic assays for the corresponding autoantibody, and for therapeutic treatment of animals including humans.
  • the present inventors have undertaken experiments designed to elucidate the regulatory mechanisms which control expression of the GAP-43 gene. Modulation of GAP-43 expression offers a convenient and effective manner in which mammals, including humans, suffering from damaged, diseased or dysfunctioning central or peripheral nervous tissue, may be therapeutically treated. Further, methods of modulating structural remodeling in normal central or peripheral nervous tissue in mammals, including humans, according to the present invention, will be a significant aid to those of skilHn further elucidating the mechanisms of neuron structure and function.
  • the antigens, antibodies and compositions of the present invention, or their functional derivatives, are well suited for the preparation of pharmaceutical compositions.
  • the pharmaceutical compositions of the invention may be administered to any animal which may experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.
  • compositions of the present invention may be administered by any means that achieve their intended purpose.
  • administration may be byparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, orbuccal routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the new pharmaceutical preparations may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally and which can be used for the preferred type Of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.001 to about 99 percent, preferably from about 0.01 to about 95 percent of active compound(s), together with the excipient.
  • the dose ranges for the administration of the compositions of the present invention are those large enough to produce the desired effect, whereby, for example, the neoplastic tissue is reduced or eliminated or ameliorated.
  • the doses should not be so large as to cause adverse side effects, such as unwanted cross reactions anaphalactic reactions and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient. Counterindication, if any, immune tolerance and other variables will also affect the proper dosage.
  • the antibodies can be admin- istered parenterally by injection or by gradual profusion over time.
  • the antibodies of the present invention also can be administered intravenously, intraparenterally, intramuscularly or subcutaneously.
  • compositions of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or soibitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as saccharides, for example lactose or sucrose, mannitol or soibitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxyprop
  • disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl-starch, cross- linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetyl- cellulose phthalate or hydroxypr ⁇ pymethyl-cellulose phthalate, are used.
  • Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water- soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the GAP-43 antigen of the present invention is unique to neuronal cells, and thus provides a convenient and useful marker. Accordingly, antibodies directed against GAP-43 may be used in various techniques well known to those of skill, to identify neuronal cells. Moreover, the antibodies of the present invention will allow detection, determination and therapeutic treatment of neoplasias and other disorders of neuronal origin, and, as such, offer a convenient and useful diagnostic and therapeutic method in vivo, in vitro or in situ, for preclinical and clinical evaluation and treatment of cancer and other disorders in animals including humans.
  • the antigen of the invention may be isolated in substantially pure form employing the antibodies of the present invention.
  • an embodiment of the present invention provides for substantially pure antigen GAP-43, said antigen characterized in that it is recognized by and binds to the antibodies of the present invention.
  • the present invention provides a method of isolating or purifying the GAP-43 antigen, by forming a complex of said antigen with one or more antibodies directed against GAP-43.
  • the substantially pure antigen GAP-43 of the present invention mayin turn be used to detect or measure antibody to GAP-43 in a sample, such as cerebrospinal fluid, serum or urine.
  • one embodiment of the present invention comprises a method of detecting the presence or amount of antibody to GAP-43 antigen in a sample, comprising contacting said sample containing said antibody to GAP-43 antigen with detectably labeled GAP-43, and detecting said label.
  • immunoreactive fractions and immunoreactive analogues of GAP-43 also may be used.
  • immunoreactive fraction is intended any portion of the GAP-43 antigen which demonstrates an equivalent immune response to an antibody directed against GAP-43.
  • immunoreactive analogue is intended a protein which differs from the GAP-43 protein by one or more amino acids, but which demonstrates an equivalent immunoresponse to an antibody of the invention.
  • GAP-43 protein contains a novel membrane-targeting peptide domain which directs the GAP-43 protein to the cell membrane, and especially to the region of the growth cone of neuronal cells.
  • the structure of this membrane- taigeting domain has been determined, and it has been shown that the peptide is effective in directing normally cytosolic proteins (which are not normally membrane-associated), to the cell membrane.
  • compositions and methods of this aspect of the invention it is possible, inter alia, to direct any desired protein to the cell membrane, including proteins which are not normally membrane-associated. Further, the compositions and methods of this aspect of the invention are of obvious utility in the therapeutic treatment of neurological damage and disorders in vitro, in vivo, and in situ, in animals. Those of skill will appreciate that the proceeding description of diagnostic and therapeutic methods is equally applicable to this embodiment of the invention. Further, it will be evident that the membrane-targeting peptide of the present invention will be of use in directing any desired protein or peptide to cell membranes, and will thus be of diagnostic and therapeutic utility in non-neurological indications as well. Examples of such indications include, but are not limited to, any applications wherein the membranes of cells may play an important role, such as immunological indications.
  • a cDNA library was generated from RNA of rat dorsal root ganglia from embryonic day 17 and cloned into the lgtl 1 expression vector (Huynh et al, in "DNA Cloning A Practical Approach,” D.M. Glover, Ed. (IRL Press,
  • GAP43-2 Three presumptive GAP-43 clones were identified with the antibody to GAP-43 described by Snipes et al., Soc. Neurosd. Abstr. 72:500 (1986). The identity of the longest clone, GAP43-2, was confirmed by hybrid-selected translation (Fig. 1). GAP43-2 selected by hybridization a messenger RNA (mRNA) that directed the translation of a polypeptide that migrated in SDS-polyacrylamide gels with the expected mobility of native GAP-43, that is, a molecular size of about 43 kD. This in vitro translation product was selectively immunoprecipitated by antibody to GAP-43.
  • mRNA messenger RNA
  • the complete nucleotide sequence of G AP43-2 and the predicted amino acid sequence are shown in Fig. 2.
  • the reading frame includes the peptide fragment that was sequenced and is in the same reading frame as the b- galactosidase gene of lgtll.
  • a cDNA for rat GAP-43 was obtained independently by J.H.P. Skene and his colleagues. Copies of the sequences were exchanged.
  • the predicted amino acid sequence of the present invention agrees perfectly with that provided by Skene, and the nucleotide sequence differs at only one position in the 3' untranslated region.
  • the methionine identified as the start of the open reading frame is the first methionine after the in-frame stop codon (TAA) at nucleotide position 13 and is surrounded by eight of the nine nucleotide consensus sequences suggested by Kozak, Cell 44:283 (1986) to be the most favorable context to initiate eukaryotic transla ⁇ tion. This suggests that it is the first residue of the GAP-43 coding region.
  • the predicted composition of GAP-43 is highly polar, without evident transmembrane domains or potential N-linked glycosylation sites. This composition is compatible with the observations that GAP-43 is membrane- associated but inaccessible to antibody recognition in the absence of membrane permeabilization (Meiri et al., PNAS USA 83:3537 (1986)); thus it may be associated with the inner face of the membrane.
  • the predicted molecular size of the GAP-43 protein from the open reading frame is 24 kD, which is less than the 43 kD originally observed by Skene and Willard as the apparent molecular size of the molecule in SDS- polyacrylamide gels (Skene etal. , J. Cell Biol. 89:86 (1981)).
  • the molecular size has been uncertain because the apparent molecular size of GAP-43 depends on polyacrylamide concentration (Jacobson et al., J. Neurosd. 6:1843 (1986)), suggesting that this protein falls in the category of proteins that migrate anomalously on SDS-polyacrylamide gels (Banker et al, J. Biol.
  • GAP-43 RNA was synthesized from the cDNA in an in vitro transcription system with the use of the bacteriophage SP6 promoter, by the method of Melton et al., Nucleic Adds Res. 72:7035 (1984).
  • RNA was generated by transcribing the cDNA cut at the Sau3A site, 65 bases 3' of the end of the predicted open reading frame (Fig. 2), and a 1100-base RNA by truncating at the Hindlll site in the polylinker region at the 3' end of cDNA.
  • Both the 800-base RNA and the 1100-base RNA directed the synthesis of a polypeptide with an apparent molecular size of 40 kD when translated in vitro with reticulocyte lysate and analyzed on a 15% SDS- polyacrylamide gel.
  • the 40-kD translation product in both cases was immunoprecipitated with the antibody to GAP-43.
  • GAP-43 synthesized in vitro from newborn rat brain RNA comigrated with these translation products.
  • GAP-43 is important to the function of growth cones.
  • GAP-43 gene expression is regulated coordinately with extension of neurites, its expression was examined in PC12 cells, in which neurite outgrowth was promoted by nerve growth factor (NGF) and to a lesser extent by adenosine 3 ',5 '-monophosphate (cAMP).
  • NGF nerve growth factor
  • cAMP adenosine 3 ',5 '-monophosphate
  • GAP-43 was expressed in a neural-specific manner (Fig. 4). At all ages, the major hybridizing band of about 1500 nucleotides is visible only in the neuronal tissues. The faint, large-molecular-size bands may correspond to unspliced precursor molecules, since the present inventors have discovered that the genomic GAP-43 gene contains intronic sequences.
  • GAP-43 mRNA in neuronal tissue is probably of neural rather than glial origin, since GAP-43 is localized in neurons (Meiri et al., PNAS USA 83:3537 (1986)) and no GAP-43 RNA was detected in the glioma cell line C6.
  • the amount of GAP-43 mRNA varies with developmental stage. Peak concentrations occur in the perinatal period, with some delay in the central nervous system relative to the peripheral nervous system. The timing of expression accords well with periods of axon growth (Jacobson, Developmental Neuropathology (Plenum, New York, 1978)).
  • GAP-43 RNA in adult neural tissues is in agreement with observations that GAP-43 protein persists in adult rat cortex, albeit in significantly lower amounts than during the perinatal period (Jacobson et al., J. Neurosd. 6:1843 (1986)).
  • the persistence of GAP-43 expression suggests an ongoing role in the adult nervous system.
  • GAP-43 regulation in the adult also occurs by alterations in gene expression.
  • One model for the function of GAP-43 in the mature animal would include an ongoing role in synaptic turnover (Cotman et al, Sdence 225:1287 (1984)) and in other "plastic" changes of the nervous system, such as learning, that are accompanied by structural growth at the nerve terminal (Bailey and Chen, Sdence 220:91 (1983)).
  • EXPERIMENTAL PROCEDURES Tissue procurement Human brain tissue was harvested fresh at the time of autopsy and within 10 hours of death. Sections of brain no larger than 2x2x0.5 cm, obtained from specific regions were snap-frozen in isopentane (2-methyl butane) cooled with dry ice and then stored at -90 C C. These specimens were used for in situ hybridization and immunocytochemistry. In addition, a small portion of tissue from the same regions, obtained fresh or from the frozen sample, was used for Northern blot analysis. Routine histopathology was performed on formalin-fixed, paraffin-embedded tissue immediately adjacent to the frozen blocks.
  • the human GAP-43 cDNA was isolated from cDNA libraries of brainstem of a 1 day old and cerebellum of a 7 year old (both libraries were from American Type Culture Collection). These libraries were screened with 32 P-labeled rat GAP-43 cDNA probes (Karns et al, Sdence 236:597 (1987)). Hybridization was for 16 hr at 42°C in 4x Standard Saline Citrate (SSC), 0.8x
  • Human GAP-43 is homologous to a mouse calmodulin binding protein
  • the cDNA for rat GAP-43 was cloned and used as a probe to identify related cDNA clones from human brainstem and cerebellum libraries. Overlapping clones were obtained from each of the libraries, which were identical in the overlapping regions.
  • the sequence of the longest clone (Cla from the cerebellum library) is presented in Figure 5A, and a comparison with rat GAP-43 shown in Figure 5B.
  • P-57 has been described as a neural- specific calmodulin-binding protein with the unusual property that it releases, rather than binds, calmodulin as calcium levels rise (Andreasen et al, Bio ⁇ chemistry 22:4615 (1983)).
  • the proteins are highly conserved between human, rat, and mouse. For example, there is 89% identity of amino acids between human and mouse. Additionally there is an unusually high degree of conservation (80%) between 3 '-untranslated regions, including 2 energetically favorable stem-loop structures shown in Figure 5C, which may, by analogy to other genes, serve to regulate messenger RNA stability (Reeves et al, Proc. Natl. Acad. Sd. USA 83:3228 (1986); Shaw et al. , Cell 46:659 (1986)). GAP-43 expression persists in discrete regions of the adult human brain
  • RNA degradation To minimize RNA degradation, brain tissue was obtained from patients with a postmortem interval of less than 10 hours. Adjacent sections were examined histopathologically. In two infants, 8 days and 1 month old, GAP-
  • GAP-43 was uniformly and robustly expressed throughout the brain as assessed by Northern blots.
  • levels in the spinal cord were low at these ages, which may be related to earlier maturation of this region (Anand et al., NewEngl. J. Med. 317:1321 (1987)).
  • GAP-43 was not expressed in any non-neuronal tissues examined either in the newborn or adult (including kidney, lung, liver, and adrenal). In the normal adult brain, GAP- 43 expression varied markedly among different regions.
  • the infarct was in the visual cortex (Area 17), and in the other, parietal lobe (Area 3,1,2,5).
  • the tissue utilized for Northern analysis and in situ hybridization included both the infarcted tissue and surrounding normal brain from the same Broadman's areas.
  • Figure 7A shows that after a stroke in Area 17, GAP-43 expression is increased to levels comparable to Area 11, the region normally most enriched in GAP-43 in the adult
  • Figure 7B shows that GAP-43 levels in Area 3,1,2,5 from two normal brains were low (lanes 1 and 2) compared to another patient with a small stroke in that location (lane 3).
  • GAP-43 is neuron-restricted in its expression, and since neurons were absent in the infarcted regions ( Figure 8A1, B), most likely the heightened GAP-43 expression derived from the morphologically uninjured neurons.
  • in situ hybridization was used to study the distribution of GAP-43 expression in the region of infarction.
  • Purkinje cells were hydropic and achromasic. As shown in Figure 9B, there was a striking enhancement of GAP-43 expression in the cerebellum, primarily in the Purkinje cell layer, a region found to be without detectable GAP-43 expression by in situ hybridization in the normal adult ( Figure 9A).
  • Growth cones are nerve terminal structures shared by developing and regenerating nerves (e.g., Ramon etal, Degeneration and Regeneration of the Nervous System, Oxford University Press, London (1928); Kater et at.,
  • GAP- 43 is one of the rapidly transported proteins which is notable for pronounced enrichment in axonal transport in developing and regenerating nerves.
  • the failure of mammalian CNS neurons to regenerate has been linked to the low and uninducible levels of GAP-43 in adult brain (Skene, Cell 37:697 (1984)).
  • it is naturally of special interest to investigate regulation of this protein in the human because of the problems encountered in treatment of CNS injury and stroke.
  • GAP-43 and the growth cone GAP-43 is highly conserved between rat and human and clearly identical to a mouse protein recently identified as a calmodulin-binding protein which has the unusual property of releasing calmodulin when ambient calcium increases (Andreasen et al., Biochemistry 22:4615 (1983); Cimler et al., J. Biol. Chem. 260:10784 (1985); Alexander et al., J. Biol. Chem. 292:6108 (1987)).
  • one notion for its role in the growth cone might be that it regulates calmodulin activity, and that it does so by releasing it in focal cellular domains.
  • GAP-43 expression is highly regulated during development. -In general, the highest levels correlate well with the periods of peak axonal elongation. However, its high level of expression in particular regions of the mature brain suggests that GAP-43 has an ongoing role in some adult neurons.
  • GAP-43 expression denotes cells actively engaged in remodeling their structure, especially at nerve terminals. Evidence for this is that in the rat the adult neurons which express GAP-43 include most prominently hippocampal neurons and mitral cells of the olfactory bulb, neurons which do in fact remodel their terminals in the adult.
  • the human hippocampus expresses GAP-43 only at low levels, suggesting that, if GAP-43 is indeed an indicator of such structural remodeling, different regions of the human brain have retained this function.
  • GAP-43 The other function proposed for GAP-43 is learning, because its phosphorylation state changes in the wake of long-term potentiation (Nelson et al., Exp. Neurol. 89:213 (1985)).
  • structural remodeling may be a facet of long-term learning (Chang et al., Brain Res. 309:35 (1984); Goelet et al., Nature 322:419 (1983)), and growth of nerve terminal areas has been documented to accompany long-term learning in Aplysia (Bailey and Chan, Sdence 220:91 (1983)).
  • neurons of the adult human brain that use structural plasticity for long-term learning are those that express GAP-43. This possibility has been discussed by several investigators, including Nelson et al, Exp. Neurol.
  • GAP-43 has been implicated because its levels closely parallel normal outgrowth and regenerative capacity (Skene, Cell 37:697 (1984)).
  • the present inventors have shown that mature neurons can express GAP-43 at high levels after certain types of stimulation.
  • Diseases such as transient ischemia and cerebral infarction are of interest because clinical recovery occurs frequently after such lesions, whereas after axotomy the impairment is permanent.
  • neurological recovery derives from repair of injured cells or sprouting of neighboring uninjured ones cannot be distinguished by clinicopathologic correlation.
  • the distance between the neurons expressing high levels of GAP-43 and the area of cell death suggests that sprouting may be involved.
  • the increased GAP-43 observed in these cases may have derived from ischemic effects upon the soma; effects which are less likely to occur during axotomy.
  • these might include the release of excitatory amino acids and consequent N- methyl-D-aspartate (NMD A) receptor excitation (Olney, in: Experimental and Clinical Neurotoxicology, P.S. Spencer and H.H. Schaumberg, eds. (Baltimore, MD: Williams and Wilkins), pp. 272-294 (1980)); Rothman, /. Neurosd. 4:1884 (1984)).
  • NMD A N- methyl-D-aspartate
  • Tissues used for in situ hybridization and immunocytochemistry were obtained fresh and snap frozen in 2-methyl butane cooled with dry ice.
  • Cryostat sections were fixed with the appropriate fixative immediately prior to use. Brain and spinal cord from embryonic (E) (days 12, 15, 18, and 20), postnatally developing (P) (days 1, 7 and 14), and adult rats were studied simultaneously. The tissue was fixed in 4% paraformaldehyde, treated with 0.3% Triton X-100 followed by 1 mg/ml proteinase K, acetylated, and pre- hybridized in 50% formamide/2x standard saline citrate (SSC). The probe was 1121 bases of GAP-43 antisense RNA, as described above.
  • Hybridization using 2 x 106 qpm per slide of ⁇ S-labeled antisense or sense riboprobe was performed in a humidified chamber for 5 hours at 50°C. The tissue sections were then washed in 2x SSC with 10 mM dithiothreitol initially containing
  • RNAase A Single stranded RNA was removed by treatment with 50 mg/ml RNAase A. The sections were further washed in 2x SSC with 1 mM DTT for 2 hours, _then dehydrated through graded alcohols containing 0.3 M ammonium acetate. The radioactive signal was detected using NTB2 Kodak emulsion. Emulsion coated slides were counterstained with hematoxylin and eosin.
  • the Western blots were developed using an alkaline phosphatase/BCIP/NBT kit (Promega). Specificity of antibody binding in the Western blot assay was demonstrated by preabsorption with GAP-43 protein purified from neonatal rat brain (Chan et al., J. Neurosd. 6:3618-3627 (1986)). GAP-43 protein was demonstrated in CNS tissue by immunohistochemical staining using the avidin- biotin horseradish peroxidase complex method (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) with 3-3' diaminobenzidine as the chromagen and hematoxylin as a counterstain. Specificity of labeling was confirmed by pre-incubation of the primary antibody with native GAP-43 protein purified from neonatal rat brain.
  • GAP-43 was expressed in relatively few neurons such that throughout most of the cerebral cortex, only scattered cells were labeled, and the intensity of labeling was markedly reduced even compared to
  • the entorhinal cortex had moderately high densities of GAP-43-expressing neurons. While dense focal labeling of neurons was still present in the spinal cord, brainstem, and cerebellum of P14 rats, in adults GAP-43-expressing neurons were either absent, or present in very low densities in these regions. However, in the adult, intense labeling of most neurons persisted in two areas: the hippocampus and olfactory bulb. The pattern of labeling in the hippocampus indicated that neurons throughout the dentate gyrus, CA1, and CA3 expressed GAP-43 mRNA. In the olfactory bulb, it was primarily the mitral cell region that contained high levels of GAP-43 mRNA.
  • GAP-43 immunoreactivity diminished in most areas, thereby leaving a more restricted distribution of GAP-43 immunoreactivity.
  • GAP-43 immunoreactivity diminished in most areas, thereby leaving a more restricted distribution of GAP-43 immunoreactivity.
  • widespread but faint neuritic labeling was evident throughout the CNS.
  • Neuronal perikarya were labeled heavily in the same regions identified by in situ hybridization as expressing high levels of GAP-43.
  • GAP-43 isolated as described herein, ⁇ -galactosidase preabsorption did not affect the labeling. There were no or few immunolabeled cells in the cerebellum, brainstem, and spinal cord, low densities of immunolabeled cells in the frontal cortex, somatosensory cortex, visual cortex, and basal ganglia, and moderately high densities in the entorhinal cortex. As noted for the in situ hybridization, intense perikaryal staining for immunoreactive GAP-43 persisted most notably in two regions: the hippocampus and olfactory bulb. Both pyramidal and granular cells throughout CAl , CA3 and the dentate gyras were labeled. In the olfactory bulb, labeling was largely restricted to the mitral cells and to neurites among the granule cells. Thus the pattern of GAP-43 immunoreactivity mirrored that of the in situ hybridization. DISCUSSION
  • GAP-43 is expressed in all CNS neurons during the perinatal period. As development proceeds, its anatomical distribution becomes progressively restricted, such that, in the adult, GAP-43- containing neurons are inhomogeneously distributed, with the highest level expression largely limited to two discrete regions: the hippocampus and olfactory bulb.
  • the antibody generated by the present inventors to the ⁇ -galactosidase- GAP-43 fusion protein permitted intense labeling of neuronal perikarya. This difference from prior reports may be due to the chimeric nature of the antigen, which perhaps exposes some different epitopes to different degrees. Alternatively, the difference may reflect omission of aldehyde fixation, which was noted by the presnet inventors to diminish perikaryal labeling.
  • the procedure of the present invention allowed the present inventors to document that the site of GAP-43 gene expression mirrored that of the GAP-43 immunoreactivity.
  • the distribution of GAP-43 in the CNS differs between rats and humans. In the adult human brain, high levels persist in associative cortical regions more than in the hippocampus (Neve et al., Proc. Natl Acad. Sci. USA 85:3638-3642 (1988)); whereas in adult rats the highest levels of GAP-43 expression are in the hippocampus, olfactory bulb, and entorhinal cortex. The significance of this finding is unclear, but it may be related to species differences with respect to regional retention of neuronal plasticity.
  • GAP-43 is expressed in cells involved in structural remodeling of synapses. Growth cones persist in certain regions of the adult brain (Sotelo et al, Lab. Invest. 25:653-671 (1971)) and direct visualization reveals ongoing synaptic rearrangements of single cells, at least in the peripheral nervous system (Purves et al., Nature 375:404-406 (1985)). In fact, there is evidence that such neuronal remodeling is integral to long- term learning (Chang et al., Brain Res.
  • GAP-43 expression is high in both the olfactory nerve and its target, the mitral cells of the olfactory bulb. Since olfactory neurons continue a cycle of death and replacement throughout life (Graziadei et al., In: M. Jacobson (Ed.), Handbook of Sensory Physiology, Vol IX, Development of Sensory Systems, Berlin, Springer-Verlag pp. 55-83 (1978)), these synapses must be continuously changing. Entorhinal neurons, which express GAP-43 in the adult, can expand their peripheral fields by sprouting into denervated zones, although it is not clear that they remodel in the absence of injury. Finally, circuitry of the hippocampus is functionally plastic (Benowitz et al., J.
  • the phenotype of an individual neuron depends upon its microenvironment.
  • Such "plasticity” is manifest, for example, in the choice of cell fate by precursor cells of the sympathoadrenal system, which assume a neuronal phenotype under the influence of nerve growth factor (NGF) or an endocrine, chromaffin cell phenotype in the presence of corticosteroids.
  • NGF nerve growth factor
  • chromaffin cell phenotype in the presence of corticosteroids.
  • Neurons additionally remodel their connections, a phenomenon termed synaptic plasticity, during normal development and in response to synaptic use (Easter et al, Science 230:507-511 (1985)).
  • Corticosteroids are necessary for normal development of the mammalian nervous system, influencing cell fate and neuronal structure and integrity (Doupe et al., J. Neurosd. 5:2119-2142 (1985); Doupe et al., J. Neurosd. 5:2143-2160 (1985); Anderson et al, Cell 47:1079-1090 (1986); Bohn et al, Dev. Neurosd. 7:250-266 (1978); Scheff et al, Expt. Neurology 68:195-201 (1980); Scheff et al., Expt. Neurology 76:644-654 (1982); Sapolsky et al, J. Neurosd. 5:1222-1227 (1985)).
  • neural crest lineage including small intensely fluorescent (SIF) cells and adrenal medullary cells, may exhibit either neuronal or chromaffin phenotypes.
  • SIF small intensely fluorescent
  • adrenal medullary cells may exhibit either neuronal or chromaffin phenotypes.
  • Corticosteroids cause them to assume chromaffin characteristics.
  • NGF neuronal growth factor
  • corticosteroids are powerful negative regulators of GAP-43 gene expression in both PC 12 cells and cultured sympathetic neurons. Further, it has been discovered that corticosteroids inhibit the stimulatory effect of NGF on GAP-43 expression.
  • Enzymes were purchased from Boehringer Mannheim, New England Biolabs, or Bethesda Research Laboratories, and used as specified by the supplier. Tissue culture products were bought from Gibco. Radiochemicais were purchased from New England Nuclear-Du Pont. Agarose and cesium chloride were purchased from Bethesda Research Laboratories. Timed pregnant Sprague-Dawley rats were purchased from Charles River Rat. Steroids were bought from Sigma and NGF from Collaborative Research (2.5s form). All other chemicals were of the highest grade available. Cell Culture
  • PC12 cells were grown in Dulbecco's modified Eagles medium (DMEM) with 5% heat-inactivated horse serum and 10% fetal calf serum. Cells were used routinely when at approximately 20% confluence. Cortisol levels, determined by RIA, were 3 nM or less in the serum-containing medium. Cells were grown in a humidified incubator with 5 % carbon dioxide at 37°C.
  • DMEM Dulbecco's modified Eagles medium
  • Cortisol levels determined by RIA, were 3 nM or less in the serum-containing medium.
  • Dissociated neurons from embryonic day 20 rat superior cervical ganglia were cultured in Ham's F12 medium supplemented with NGF (50 ng/ml), 0.6% glucose and 10% fetal calf serum.
  • NGF 50 ng/ml
  • glucose 0.6%
  • fetal calf serum 10%
  • the compounds were usually dissolved in 95% ethanol.
  • Controls performed with ethanol as a vehicle revealed no change in the abundance of GAP-43 or GAPDH RNA.
  • Total RNA was prepared from cultured cells by the guanidine isothiocyanate method (Chirgwin et al., Biochemistry 78:5294-5299 (1979)). Twenty micrograms of total RNA from each culture were electrophoresed through a 1.2% agarose gel containing 2.2 M formaldehyde, transferred by capillarity to Nytran (Schleicher and Schuell), and the nucleic acid immobilized by heat fixation.
  • Prehybridization was done for at least 1 hour in a hybridization solution containing 50% formamide, 5xSSC (lxSSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), lx Denhardt's solution, 1% sodium dodecylsulfate (SDS) and 100 mg/ml denatured salmon sperm DNA at 42°C.
  • 5xSSC lxSSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0
  • SDS sodium dodecylsulfate
  • Hybridization was performed for 10-12 hours at 42°C in the same solution, containing 1x105 cpm ml of 32 P-labeled DNA probe prepared by random hexanucleotide priming using the Klenow fragment of DNA polymerase I (Feinberg et al, Anal. Biochem. 732:6-12 (1984)).
  • the probes were made from cloned cDNAs for GAP-43 as described herein or for
  • Blots were stripped of hybridized probe at 80°C for 2 hours in a solution containing lx Denhardt's solution, 1 % SDS, 50 mM tris, pH 7.4, and 0.05% sodium pyrophosphate.
  • RNAsin 200 units/ml RNAsin (Promega Biotec). Nuclei were resuspended after washing with lysis buffer in 50 mM Tris, pH 8.3, 40% glycerol, 5 mM magnesium chloride, 0.1 mM ethylenediamine tetraacetic acid (EDTA), 2mM dithiothreitol and 200 units/ml RNAsin. Nuclei were counted and stored in
  • Labeling of nascent chains in thawed nuclei was performed by adding to the suspended nuclei an equal volume of buffer containing 10 mM Tris, pH 8.0, 5 mM magnesium chloride, 0.3 M potassium chloride, and 10 mM each of adenosine, cytidine and guanosine nucleotide triphosphates. Three hundred mCi of ( ⁇ P) uridine triphosphate were then added (3000 Ci/mmol), and the nuclei labeled for 30 minutes at 30°C.
  • Nuclei were then digested with 100 units RQ1 DNAase (Promega Biotec) added in 600 ml of a buffer containing 60 mM Tris, pH 7.5, 15 mM sodium chloride, 10 mM magnesium chloride, and 200 units/ml RNAsin for 45 minutes at 37 °C.
  • the labeled RNA was then digested with proteinase K (Boehringer Mannheim) as described (Green ⁇ berg et al., J. Biol. Chem. 260:14101-14110 (1985)).
  • nucleic acids were ethanol-precipitated in the presence of sodium acetate.
  • the recovered labeled nucleic acid, which still contained DNA was subjected to another cycle of RQ1 DNAase (50 units enzyme in 250 ml buffer containing 50 mM Tris, pH 7.5, 10 mM sodium chloride, 7.5 mM magnesium chloride and 200 units RNAsin ml) for 45 minutes at 37°C, and then proteinase K (by adding 100 ml of buffer containing 5 % SDS, 0.5M Tris, pH 7.4, 125 mM EDTA and 0.2 mg ml proteinase K) for 30 minutes at 42°C.
  • RQ1 DNAase 50 units enzyme in 250 ml buffer containing 50 mM Tris, pH 7.5, 10 mM sodium chloride, 7.5 mM magnesium chloride and 200 units RNAsin ml
  • proteinase K by adding 100 ml of buffer containing 5 % SDS, 0.5
  • RNA was subjected to three cycles of ethanol precipitation with ammonium acetate to remove unincorporated nucleotide triphosphates.
  • Plasmids containing cloned cDNAs for tyrosine hydroxylase (Lewis et al., J. Biol. Chem. 285:14632-14637 (1983)), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pBR322, and an 8 kilobase genomic fragment of the GAP-43 gene, were linearized with appropriate restriction enzymes, phenol extracted, ethanol precipitated and recovered by centrifugation.
  • DNA 250 mg/ml
  • Prehybridization of the filters was done in a buffer containing 25 mM sodium PIPES, pH 7.2, 50% formamide, 0.75 M sodium chloride, 2.5 mM EDTA and 100 mg/ml of tRNA at 45°C for 10-12 hours.
  • Hybridization was performed in the same buffer containing labeled RNA at specific activities ranging from 1-3 x 106 cpm/ml for 4 days at 45 °C. Washing and RNAase treatment were performed as described (Greenberg et al., J. Biol Chem. 260:14101-14110 (1985)). Filters done in duplicate were counted after drying in a scintillation counter. Data are expressed as parts per million hybridized after subtracting background from vector containing filters.
  • NGF and glucocorticoids upon GAP-43 mRNA accumulation were measured by RNA blotting.
  • NGF addition resulted in a marked increase over the basal level, whereas dexamethasone caused a prominent diminution in GAP-43 mRNA levels.
  • Quantitation of GAP-43 RNA revealed that NGF caused a 3.5 fold increase, while dexamethasone lead to a 5.5 fold decrease.
  • Accumulation of GAP-43 mRNA in the presence of NGF was persistent, unlike that of c-fos (Greenberg et al, J. Biol Chem. 260:14101- 14110 (1985)), which peaks within several hours and then rapidly declines despite the continued presence of NGF.
  • steroid concentration was 1 mM for 48 hours of treatment.
  • the quantitation was derived by densitometry and normalized for slight variations in RNA input.
  • Estradiol, testosterone, and pregnenolone had no effect on accumulated GAP-43 mRNA levels.
  • Dexamethasone, cortico- sterone, aldosterone and progesterone reduced the levels of GAP-43 RNA to 6%, 15%, 10% and 15% of control (defining the NGF-stimulated level of GAP-43 RNA as 100%), respectively.
  • These data suggest activation of either the mineralocorticoid or glucocorticoid receptors, although the progesterone effect may be mediated by its own receptor (Arriza et al., Sdence 237:268-
  • Corticosteroids block the NGF induction of a neuronal phenotype in both SIF and adrenal medullary cells (Doupe et al, J. Neurosd. 5:2119-2142 (1985); Doupe et al., J. Neurosd. 5:2143-2160 (1985)).
  • PC12 cells were grown for 36 hours in the presence of NGF (50 ng/ml) and dexamethasone (1 mM). The results indicate that dexamethasone prevents the NGF-mediated increase in GAP-43 mRNA.
  • Corticosteroids suppress GAP-43 in sympathetic neurons
  • NGF-treated PC12 cells are considered good models of differentiated neurons, it was desired to determine whether the effects of corticosteroids might be exerted upon primary neurons after they had achieved their fully differentiated state.
  • dissociated neurons of the rat superior cervical ganglion were cultured, to which 1 mM dexamethasone was added for 48 hours.
  • Total RNA was prepared, fractionated, blotted and probed as before.
  • Dexamethasone reduced the expression of GAP-43 RNA in sympathetic neurons.
  • the morphological appearance of the neurons in the dexamethasone-treated cultures was not different than that of the untreated cells. This suggests that neurite extension or maintenance over the short term may not depend upon the persistence of GAP-43 mRNA, but that long-term effects require evaluation since the GAP-43 gene product may have a long half-life.
  • GAP-43 gene expression is subject to both positive and negative control: positive by NGF and negative by glucocorticoids. Both effects are direct, neither requiring new protein synthesis.
  • cycloheximide was shown to further augment the dexamethasone suppression of GAP-43 mRNA. While not intending to be bound by a particular theory, this may be due to inhibition of synthesis on an mRNA-stabilizing protein.
  • the GAP-43 gene is highly regulated. To-date it has been reported only in neurons. The present inventors, however, have obtained data which support low level expression in other cells that derive from the neural crest. Peak levels in the animal are achieved at the time of neurite growth, relating either to normal development or to regeneration. The molecular regulators of its cell-specific and growth-related expression have not yet been elucidated. Nerve growth factor directly increases expression of several genes, such as c-fos, NGFIA, NGFIB, beta actin and a cloned cDNA related to intermediate filaments (Greenberg et al., J. Biol. Chem.
  • steroids act through their receptors as transcriptional regulators, it is important to determine which neuronal genes are regulated by corticosteroids. In considering cells of neural crest lineage, it is of particular interest to determine whether the antagonistic effects of corticosteroids and NGF on cell phenotype are mirrored at the level of gene expression, i.e., whether the same gene may be bimodally regulated by the two agents.
  • GAP-43 transcription is suppressed by corticosteroids, and that the concomitant presence of NGF does not prevent this suppression. This is similar to the glucocorticoid inhibitory effect on NGF-mediated neuronal differentiation of cultured chromaffin cells (Doupe et al, J. Neurosd. 5:2119-2142 (1985)).
  • GAP-43 is dually regulated by NGF and corticosteroids in a manner at least compatible with the known divergent effects of these modulators of cell pe.
  • SCG 10 another neural-specific gene, designated SCG 10
  • SCG 10 gene expression is stimulated by NGF and repressed by gluco- corticoids, although the levels of control differ somewhat between the two genes.
  • the protein may have a long cellular half-life. Additionally, PC12 cells are transformed and likely subject to different structural constraints from those exerted upon their in vivo counterparts.
  • GAP-43 might contribute directly to the establishment of the unique neuronal phenotype, perhaps at the level of the cytostructure.
  • expression vectors encoding rat GAP-43 are introduced into several types of non-neuronal cells. Expression vectors were constructed using rat GAP-43 cDNA (Karns, et al., Sdence 236:597 (1987)) inserted into plasmids containing the SV40 origin of replication under the control of the adenovirus major late promoter, the SV40 early promoter, or the cytomegalovirus promoter. The results were similar using all of these vectors.
  • COS 7 cells (Gluzman, Cell 23:175 (1981)) were transfected as described (Zuber, etal., Sdence 234:1258 (1986)) and examined for GAP-43 immunoreactivity using rabbit anti-GAP antibody as described above. Control transfections were done identically using a similar vector expressing the T-cell-specific membrane protein CD8 (Seed, et al., Proc. Natl. Acad. Sd. USA 84:3365 (1987)). Cells were examined 1 hour after plating.
  • Control COS cells were essentially round after immunofluorescent labeling of the CD8-transfected cells with antibody to CD8.
  • GAP-43 transfected cells GAP-43 immunoreactivity was prominent in about 5-20% of the cells, depending upon transfection efficiency.
  • GAP-43 expression clearly correlated with process extension. Many cells expressing GAP-43 extended filopodial processes that were narrow and between 20 and 75 mm in length. In both control and cell lines expressing GAP-43, the perimeter often included broad, thin, raffled lamellipodia.
  • Process formation was assessed from four independent lines transfected with a control plasmid and four independent lines expressing the highest amounts of GAP-43, as determined by Western blot. Living cells were examined by the use of Nomarski optics. All CHO cell lines with GAP-43 immunoreactivity had a greater tendency to extend processes than did control cell lines. In addition, cells expressing GAP-43 often had multiple processes (ranging between 6% and 11% of cells) as compared to control cell lines (from 0.5% to 1%), and the process length was longer than in the control cells. The neuronal protein GAP-43 therefore causes a change in the shape of these non-neuronal cells. Filopodia and lamellipodia extend directly from the cell soma, such that the cell protrusions resemble growth cones.
  • GAP-43 In non-neuronal cells, GAP-43 is removed from its normal biological context, and is expressed in a deregulated fashion, so the changes observed here may not mimic the effect of GAP-43 in its neuronal context.
  • GAP-43 is, in fact, related to growth cone function, in that it is enriched in growth cones (Katz, et al, J. Neurosd. 5:1402 (1985); DeGraan, et al., Neurosd. 61:235 (1985); Meiri, et al, Proc. Natl. Acad. Sd.
  • GAP-43 a neuron-specific molecule, is able to contribute a completely novel and neuron-like structure to these cells. Another explanation is that GAP-43 interacts with more general mechanisms that control cell shape (Bray, et al., Sdence 239:883 (1988); Smith, Sdence 242:708 (1988)).
  • growth cone structure has been suggested to utilize cellular mechanisms, such as flow of cortical actin and selective adhesion, that may be used as a general means to impart cellular motion (Bray, etal., Sdence 239:883 (1988); Smith, Sdence 242:708 (1988)). How GAP-43 might interact with such machinery remains to be determined.
  • GAP-43 protein contains a novel membrane-targeting peptide domain which directs the GAP-43 protein to the cell membrane, and especially to the region of the growth cone of neuronal cells.
  • the structure of this membrane- targeting domain has been determined, and it has been shown that the peptide is effective in directing normally cytosolic proteins (which are not normally membrane-associated), to the cell membrane.
  • compositions and methods of this aspect of the invention it is possible, inter alia, to direct any desired protein to the cell membrane, including proteins which are not normally membrane-associated. Further, the compositions and methods of this aspect of the invention are of obvious utility in the therapeutic treatment of neurological damage and disorders in vitro, in vivo, and in situ, in animals.
  • GAP-43 while it is associated with cell membranes, and especially with the growth cones of developing or regenerating neuronal cells, lacks any such highly hydrophobic region. It has now been discovered that the GAP-43 protein is encoded in three exons, as shown in Figure 2. The short (10 amino acid residues) amino- terminus exon has su ⁇ risingly been discovered to encode a membrane- targeting peptide domain. Experiments in which large portions of the second GAP-43 exon were removed did not affect membrane binding of the remaining protein. Similarly, it was found that replacing the carboxy-terminus of GAP-
  • membrane-targeting peptide any amino acid sequence as follows:
  • the membrane-targeting peptide of the invention may be attached to a desired protein or peptide by well known methods, including but not limited to direct synthesis by manual or, preferably, automated methods.
  • An alternate preferred method by which the membrane-targeting peptide of the invention may be attached to the desired protein or peptide involves modifying the gene encoding the desired protein or peptide, so that the expressed gene product will include the membrane-targeting peptide at its amino-terminus end. This may be accomplished by well-known methods, including but not limited to blunt-ended or sticky-ended ligation methods as described herein.
  • the present invention provides for cDNA coding for a membrane-targeting domain comprising the nucleotide sequence
  • non-neuronal cells including COS cells, NIH 3T3 cells, and CHO cells
  • neuronal cells PC12 cells
  • plasmids containing the GAP-43 gene in which mutations were introduced into the nucleotide sequence of the cysteines at positions three (C3) or four (C4), to result instead in expression of alanine at those positions ( Figure 11).
  • the invention comprises an amino acid sequence comprising
  • peptides including the first five, six, seven, eight or nine amino acids of exon 1 also will allow membrane binding when attached to a desired protein or peptide.
  • Those of skill will appreciate that the sufficiency of these intermediate length peptides for directing membrane binding in particular applications may be determined by the exercise of merely routine skill, with the benefit of the teaching of the present invention. Accordingly, the same and their equivalents are to be considered as within the contemplated scope of the present invention.
  • the first GAP-43 exon described above was ligated at the amino-terminus end of the gene encoding chloramphenicol acetyl transferase (CAT), a protein which is normally cytosolic, and not membrane-associated.
  • CAT chloramphenicol acetyl transferase
  • GAP-43 will direct CAT to the same location as GAP-43 in transfected PC12 cells. These cells resemble neuronal cells in putting out long processes tipped by growth cones. GAP-43 is normally especially enriched in neuronal cell growth cones, and data suggest that the membrane-targeting peptides of the present invention are responsible for this observed growth cone enrichment.
  • the present inventors employed mutational analysis and laser scanning confocal microscopy of fusion proteins that included regions of GAP-43 and chloramphenicol acetyltransferase (CAT). .It has consequently been verified that a short stretch of the GAP-43 amino terminus suffices to direct accumulation in growth cone membranes, especially in the filopodia.
  • CAT chloramphenicol acetyltransferase
  • Constructions that encoded varying amounts of the GAP-43 amino terminus fused to a reporter peptide were expressed in COS and PC 12 cells. Chloramphenicol acetyl transferase (CAT) was chosen as the reporter peptide because it is cytosolic when expressed in eukaryotic cells and is very stable. Plasmids were constructed that encode fusion proteins of the first 10 amino acids of GAP-43, MLCCMRRTKQ, fused to the amino terminus to the complete CAT protein (GAP10CAT), or the first 40 amino acids of GAP-43 fused to CAT (GAP40CAT).
  • CAT Chloramphenicol acetyl transferase
  • Immunoblotting was carried out as follows: Chimeric proteins with the amino terminus of GAP-43 fused to CAT associate with COS cell membranes. CAT, GAPIOCAT and GAP40CAT were transiently expressed in COS cells.
  • Immunoblots of membrane (M) and cytosolic (C) fractions from each transfec ⁇ tion were prepared using anti-CAT antibody.
  • CAT-transfected cells immunoreactivity is found only in the cytosolic fraction and co-migrates with purified CAT protein.
  • GAP40CAT and GAPIOCAT cells nearly all of the immunoreactivity is membrane-associated and migrates more slowly than CAT, as expected for fusion proteins with Mr 4000 or 1000 greater than CAT.
  • Molecular weight standards of 116, 84, 58, 48.5, 36.5 and 26.6 kilodaltons were used.
  • Membrane association of GAP and GAP40CAT was evaluated in PC12 cells. Stably transfected PC12 cells expressing CAT, GAP40CAT, or GAP were selected as described herein. Immunoblots of membrane (M) and cytosolic (C) fractions were stained with anti-CAT or anti-GAP antibodies. CAT-transfected cells (CAT) contained immunoreactivity in the cytosolic, but not in the membrane fraction, and this immunoreactive CAT co-migrated with purified CAT. In contrast, GAP40CAT transfected cells (G40CAT) contained membrane-associated CAT immunoreactivity which migrated more slowly. Fractions from rat brain (BR) demonstrated that most, but not all, endogenous GAP-43 immunoreactivity is membrane-associated. In transfected PC 12 cells over-expressing GAP-43, nearly all of the GAP-immunoreactivity is membrane-associated and co-migrates with purified GAP-43.
  • the GAP-43 coding sequence replaced the stuffer at the Xba I sites of the CDM8 plasmid described by Seed, Nature 329:840-846 (1987).
  • the inserted GAP ⁇ 43 sequence included the entire coding sequence of rat GAP-43, from the Nla III site at the start of translation to the Sau 3 Al site 68 bp downstream from the termination codon, as described herein.
  • the CAT expression plasmid pCAT, the Hind III to Bam HI fragment containing the CAT coding sequence and polyadenylation site from pSV2CAT (Gorman, et al., Mol. Cell. Biol.
  • pGAP40CAT and pGAPlOCAT include the first forty or ten amino acids of GAP-43, respectively, fused in-frame with CAT in pCAT by the use of polylinkers.
  • DEAE dextran and chloroquine was used as described (Zuber, et al. Sdence 234:1258-1261
  • PC12 cells For stable transfection of PC12 cells a neomycin resistance plasmid co-transfected with the plasmid of interest on a 1 to 10 ratio was used as described herein. During selection of PC12 cells, 400 ug ml of active Geneticin (GIBCO) were used. Transient transfection of PC 12 cells was performed by electroporation with the Bio-Rad Inc. electroporation system using 300 volts and 960 microfarad. After 8 hour the medium was changed. Twenty-four hours after electroporation the cells were plated on poly-D-lys- ine-coated coverslips in the presence of 50 ng ml NGF and analyzed 24 hours later.
  • GEBCO active Geneticin
  • rabbit anti-GAP-43 antibodies were made by immunizing rabbits against four peptides including aa 1 to 24, aa 35 to 53, aa 53 to 69, and aa 212 to 228 of rat GAP-43.
  • Anti-GAP-43 antibody was affinity-purified on GAP peptide agarose.
  • Anti-GAP antibody was bound to a resin that contained 10 mg/ml of each peptide coupled to agarose by the cyanogen bromide method and the antibody was eluted at pH 3.5.
  • Rabbit anti-CAT antibodies were obtained from 5 Prime-3 Prime, Inc. Secondary antibodies were obtained from Organon Teknika, Jackson Immunologicals, and Vector labs.
  • COS or PC12 cells were scraped from 100 mm confluent petri dishes and pelleted at 2000 x g for 10 minutes.
  • the pelleted cells were homogenized by Polytron in 10 mM Tris- HC1, ImM EDTA, pH 7.6 (300 ul/dish) and centrifuged at 250,000 x g for 30 minutes at 4°C. The supernatant was collected as the cytosol fraction.
  • the pellet was washed by homogenization and centrifugation in the same buffer, and then resuspended to the same volume as the cytosol fraction.
  • Rat brain was obtained from 1 day old rats and homogenized by Polytron in 10 mM Tris-HCl, ImM EDTA, pH 7.6 (10 ml/gram wet weight tissue). The cytosol and washed membrane fractions were prepared by centrifugation as described for the cell extracts.
  • GAP-43 protein was purified from rat brain by a modification of the method of Andreasen et al, (Biochemistry 22:4615-4618 (1982)) and used as a positive control for immunostaining.
  • the same volume of cytosol or membrane fraction (usually 100 ul) was electrophoresed on polyacrylamide gels (Laemmli, N ⁇ flire 227:680-685 (1970)). Proteins were electrophoretically transferred to nitrocellulose and excess sites were blocked with 4% BSA. Membranes were then incubated for 24 hour at 4°C with 40 ug/ml affinity purified anti-GAP, or a 1:1000 dilution of anti-CAT antibodies. Bound antibody was detected using anti-rabbit Vectastain horseradish peroxidase method according to the manufacturer's instructions. Tetramethyl benzidine
  • GAP-43 and the GAP-CAT chimeric proteins in NGF-treated transfectants of PC12 cells was investigated by confocal microscopy in order to determine whether the amino terminus accounts for the growth cone enrichment of GAP-43 in neuronal cells.
  • CAT remains cytosolic, whereas GAP-43 is distributed in a punctate pattern with notable enrichment in growth cones, a pattern similar to that of native GAP-43 in neurons.
  • the amino terminus of GAP-43 fused to CAT caused the resulting fusion protein to acquire a distribution that closely resembled that of GAP-43 itself.
  • Perinuclear labeling for both GAP-43 and the chimeric protein was detected at a low level, and may be due to localization to the Golgi, as has been observed for native GAP-43 (Van Hooff etal., J. Cell Biol. 708:1115-1125 (1989)).
  • Glutaraldehyde fixation provided better histologic preservation of the finer processes of the growth cones, and revealed that the chimeric protein accumulates especially within filopodia.
  • Subcellular localization of CAT, GAP-43 and fusion proteins in transfected PC12 cells was carried out as follows: Confocal immunofluorescence of (A) CAT, (B) GAP-43, (C) GAP40CAT, and (D) GAPIOCAT in PC12 cells revealed that CAT labeling is diffuse and cytosolic whereas GAP-43 is localized to the membrane in a punctate fashion with some enrichment in the growth cones. When either the amino terminal 40 amino acids (GAP40CAT) or 10 amino acids (GAPIOCAT) were fused to CAT, the immunofluorescent distribution resembled that for GAP-43, including enrichment in growth cones. All cells were treated with NGF for 24 hours prior to fixation.
  • Anti-CAT antibody was used for CAT, GAP40CAT and GAPIOCAT, whereas anti-GAP-43 antibody was used for GAP-43.
  • Control PC 12 cells of this variant expressed undetectable levels of GAP-43 and CAT immunoreactivity.
  • PC12 cells were transferred to poly-D-lysine coated coverslips 24 hours before immunofluorescence in the presence of 50 ug/ml nerve growth factor (NGF). Fixed with 3.7 % formaldehyde for 7 minutes, and permeabilized with 0.1 % Triton-X-100 for 3 minutes.
  • NGF nerve growth factor
  • GAP40CAT Localization of GAP40CAT within the growth cone of a PC12 cell was demonstrated using a higher power comparison of PC12 cells expressing GAP40CAT viewed with Nomarski optics and scanning confocal immunofluorescence, labeled with anti-CAT antibodies. Cells had been treated with NGF for seven days. One growth cone appeared brightly labeled, but a smaller one did not. Unequal labeling of different growth cones, even of the same cells, occurs for native GAP-43 in neurons (Goslin et al, Nature 336: 672-674 (1988)) as well. Comparison of the Nomarski and immunofluorescent images showed that filopodia were especially labeled. Similar results were seen for GAPIOCAT.
  • the growth cone membrane is also distinctive in its protein make-up.
  • One interesting possibility is that the growth cone membrane has binding sites that recognize and bind the palmitylated amino terminus of
  • GAP-43 and GAP-CAT fusion proteins bind to the membrane of non-neuronal cells, similar or identical binding sites must be present in other cell types, but because GAP-43 is neuron-specific, these sites would presumably be targets for different proteins in non-neuronal cells.
  • GAP-43 causes enrichment especially in filopodia. This is the normal location of GAP-43 in these cells, as evidenced by electron microscopy (Van Hooff, et al., J. Cell Biol.
  • the present invention provides, in another aspect, for a method of introducing a desired protein or peptide into the membrane region of a neuronal or non-neuronal cell, and for a method of directing a desired protein or peptide to the growth cone areas of neuronal cells.
  • a method for directing a desired protein or peptide to the membrane of a cell comprising
  • step (b) introducing the resulting protein or. peptide comprising said membrane-targeting domain into a cell; wherein the resulting protein or peptide of step (b) is directed to said membrane of said cell by said membrane-targeting domain.
  • the present invention provides nucleotide sequences encoding the membrane-targeting peptide comprising the above amino acid sequences or their functional or chemical derivatives, as well as the addition of these sequences by well known methods to nucleotide sequences encoding proteins or peptides other than GAP-43 (as well as GAP-43 itself), and the expression of the resulting sequences in prokaryotic or eukaryotic hosts by methods well known to those of skill.
  • the desired protein or peptide may be diagnostically or therapeutically labeled, and the utility of the composition and methods of this aspect of the invention will be apparent to those of skill, and may be readily utilized for in vitro, in vivo, or in situ diagnostic or therapeutic purposes in animals including humans with the exercise of merely routine skill.
  • GAP-43 regulation occurs at the level of gene expression. Until the present time, however, nothing has been known about cis or trans-acting elements that might regulate its expression. Naturally, it would be of great interest to define elements of the GAP-43 gene that confer its responsiveness to growth factors, cause cellular restriction of expression, and regulate the gene during development of the nervous system. In order to identify regulatory elements, the entire rat genomic DNA of GAP-43 has been cloned. Accordingly, genomic GAP-43 has been isolated, and its intron-exon boundaries and transcriptional start sites have been mapped.
  • the promoter is quite unusual in its structure, containing a repetitive sequence capable of forming unusual conformations, and lacking some canonical promoter components. Transcription can initiate from more than one site, and some of the start sites are utilized differently in the central and peripheral nervous systems.
  • the inventors have investigated whether the GAP-43 promoter contains regions recognized by brain-specific nuclear proteins. Regions of the GAP promoter have been examined by gel electrophoresis mobility shifts, and a domain which binds protein(s) present in brain but not in liver nuclear extracts has been identified. The binding activity diminishes with brain maturation.
  • the binding site is limited to a stretch of about 20 nucleotides, which also is specifically protected in DNase protection assays by brain nuclear extracts and not by liver extracts.
  • the region has a sequence similar to binding sites recognized by a class of DNA binding proteins known as POU.
  • Genomic clones containing the three GAP-43 exons were isolated from a library constructed by inserting size fractioned SauIII A partial digests of rat genomic DNA into the BamHI site of bacteriophage EMBL-3.
  • the library was initially screened on Colony Plaque Screen filters (DuPont/NEN) following standard protocols with random primed GAP-43 cDNA, as described hereinabove.
  • the library was replated and duplicate lifts were probed sequentially with three oligonucleotides complementary to the 5' most region of the cDNA (#4, -68 to -39; #2, -38 to -9 and #5, +1 to +20 in Figure 14). Clones positive for at least two oligonucleotides were selected for further analysis. Inserts from positive phage were subcloned into the Sail site of the pBluescript vector (Stratagene) for mapping with a variety of restriction enzymes.
  • the GAP-43 promoter was sequenced by the dideoxy method of Sanger et al, Proc. Natl. Acad. Sd. USA 74:5463-5467(1977) using Sequenase as described by the supplier (USB). Subclones of the bacteriophage clones containing the first exon were constructed by standard methods in pBluescript (Stratagene) for double stranded sequencing and in M13 vector (Messing,
  • RNAse protection analysis was done as described in Krieg et al., Meth. Enzymol. 155:397-415 (1987).
  • a genomic piece of GAP-43 from the Xbal site at -475 from the translation start site to the Sspl site at +83 (in the first intron) was cloned into the Xbal and EcoRV sites of pSP72 (Promega).
  • RNAse protection analysis showed three major GAP-43 transcripts at -47/48, -51/52 and -78 bases from the translational start site. Protections were performed on tRNA, RNA prepared from newborn rat lung, dorsal root ganglia, and cerebral cortex. The probe extended 475 bases upstream from the translational start site (Xbal site). An over exposure showed additional longer transcripts which were much more abundant in the cerebral cortex as opposed to the DRG. Markers were MSPII digested pBR322.
  • RNAse protection analyses were carried out showing the heterogeneity of GAP-43 transcripts in different areas of the nervous system and in PC12 cells.
  • RNA from control PC12 cells was compared with that obtained from NGF treated PC12 cells, tRNA, DRG, cerebellum, cortex, and hippocampus.
  • RNA samples derived from the CNS had a higher proportion of the longer transcripts than samples from DRG or PC 12 cells.
  • the genomic piece of GAP-43 from the Ndel site at -233 to the same Sspl site at +83 was cloned by digesting the plasmid described above with Ndel and Hindlll and filling with Klenow fragment of DNA polymerase. The Hindlll site was reformed.
  • transcripts were elongated with T7 polymerase after linearizing the vectors with Hindlll. Thus, all transcripts extending beyond this site accumulated as a single band at -234.
  • RNA samples from newborn rat heart, liver, lung, cerebellum, spinal cord, cortex, hippocampus, and dorsal root ganglia were used.
  • the longer upstream start sites as a group constituted the start sites of a significant fraction of RNA in the central nervous system tissues but not in the dorsal root ganglia.
  • a rat genomic library was screened with probes derived from the GAP-43 cDNA, as described herein above.
  • Initial screening with radiolabeled full length cDNA provided two classes of phage, which subsequent analysis showed to correspond to the second and third exons of the gene. Because the first exon proved to be small, and hence underrepresented in the cDNA probe, additional rounds of screening using three oligonucleotide probes derived from the 5' most region of the cDNA were necessary in order to obtain clones containing the 5' end of the gene.
  • a map of the GAP-43 gene is shown in Figure 13a, with represen ⁇ tations of the phage used to map it. The gene spans at least 50 kb and contains 3 small exons.
  • the first is about 80 bp (see below for a description of the variability of the 5' end), the second is 565 bp, and the third is 672 bp, and they are separated by 2 introns of greater than 24 kb and 20 kb, respectively.
  • the first exon contains the 5' untranslated sequences of the mRNA and encodes the first 10 amino acids of the protein. This short amino terminal region of the protein contains the "sorting sequence” that directs binding of GAP-43 (and heterologous fusion proteins) to growth cone membranes, as described hereinabove.
  • the second exon encodes the bulk of the protein and includes a region identified by Alexander et al, J. Biol Chem. 263:7544-7549 (1988) as the calmodulin binding site.
  • the third exon encodes the carboxy-terminal 28 amino acids and contains 587 bases of untranslated sequence and the poly-A addition site.
  • the GAP-43 promoter contains H-DNA
  • the sequence of the 5' region of the gene is displayed in Figure 14. It contains no TATA or CAAT boxes, but does contain a sequence, TATTCATG (overlined), which is identical to the consensus Pit-1 binding site. This octamer binds a class of proteins thought to regulate transcription of several genes, including prolactin and growth hormone (Bodner et al., Cell
  • a striking feature of the promoter sequence is that more than 80% of the coding strand is composed of purines (underscored by asterisks in the figure), with two uninterrupted purine homopolymer stretches spanning from -118 to -188, and from -238 to -370, respectively. Some areas of these homopolymer stretches that are not simply alternating G and A contain tandem repeats, which possess some mirror symmetry (for example -168 to -118). Hai ⁇ in forming palindromes centered at -112, -232 and -509 flank the homopolymer regions and may influence secondary structure.
  • Htun et al. Sdence 241:1791-1795 (1988) devised a simple gel system to demonstrate that H-DNA will introduce a severe kink in DNA. Their assay is based upon the enhanced stability of H-DNA at low pH. When fragments of DNA which contain an H-forming region are electrophoresed at low pH, an H-DNA induced kink will retard mobility as compared to the mobility at a pH not favoring H-DNA formation (Htun et al., Sdence 247:1791-1795 (1988)). The present inventors exploited this mobility shift to demonstrate that the purine homopolymer region from -240 to -370 in the GAP-43 promoter is capable of forming stable H-DNA structures in linear DNA in vitro.
  • Figure 15 is a representation of the restriction digest fragments of the GAP-43 promoter which were analyzed by gel electrophoresis, as described hereinbelow. The potential H-DNA forming homopurine-homopyrimidine regions are shown as thickened lines.
  • aliquots of the digests represented in Figure 15 were loaded on 1.4% agarose gels that had been equilibrated with Tris-acetate at either pH 7.4 or 4.0 and ran in parallel. Bands that shifted at pH 4 exhibited smearing that may result from the B to H transition (Htun et al., Sdence 241:1791-1795 (1988)).
  • GAP-43 upstream sequence Another notable feature of the GAP-43 upstream sequence is the absence of the TATA motif. Genes that lack a TATA sequence to direct initiation of transcription often have multiple mRNA start sites. This proved to be true for GAP-43.
  • RNAse protection analysis was used to determine the transcriptional start sites for GAP-43 in several tissues. RNA from lung, dorsal root ganglia (DRG) and cerebral cortex (CTX) was analyzed with a probe extending to -475 bases from the translation start site. Using this probe, three major bands were protected, corresponding to transcriptional start sites at -47A48, -51/-52, and -78. These same sites were identified by primer extension. Additional minor bands become visible after longer exposure. -
  • transcripts at around -230 are present to a much greater extent in mRNA from the cerebral cortex as compared to the dorsal root ganglia.
  • GAP-43 gene expression in the central and peripheral nervous system is different (Skene et al., J. Cell Biol. 89:86-95 (1981); Skene et al., J. Cell
  • RNA from other areas of the CNS was analyzed.
  • RNA from the hippocampus, cortex and cerebellum has a higher proportion of the transcripts initiating from the area around -230 than RNA from DRG or PC 12 cells, although the amount of each of these longer messages is relatively small.
  • a probe was used that pools all messages that start beyond -234, the difference between start sites in the CNS and PNS becomes more apparent.
  • the present embodiment of the invention is directed to the isolation and characterization of genomic sequences containing the GAP-43 gene.
  • Three small exons corresponding to the ⁇ 1.5 kb mRNA are separated by introns of at least 24 and 20 kb, respectively.
  • the promoter region is rather unusual. There are several long homopurine-homopyrimidine stretches in the upstream region which are potentially capable of forming triple stranded "H-DNA" (Wells et al., FASEB 7.2:2939-2949 (1988)). It is here demonstrated that one of these regions does, in fact, form H-DNA in vitro.
  • the promoter lacks a canonical TATA box, and has multiple transcription initiation sites. The utilization of some of these sites differs in various parts of the nervous system.
  • the rat GAP-43 gene is a single copy gene that consists of three exons and two introns spanning at least 50 kb.
  • the present inventors have obtained some evidence that the exons correspond to functional domains in the protein.
  • the first exon which encodes only the first 10 amino terminal residues, contains the stretch responsible for membrane targeting of GAP-43. Cysteines at positions 3 and 4 in the protein are acylated and may be involved in membrane binding, as described hereinabove. The amino terminus is -Ill-
  • the second exon includes the calmodulin binding region from amino acid 43 to 51 (Alexander et al., J. Biol. Chem. 263:7544-7549 (1988)) as well as a serine at position 41 that is a substrate for protein kinase C (Coggins et al., Soc. Neurosd. Abstract (1988)).
  • Exons I and II contain regions that are well conserved between fish and several mammalian GAP-43 proteins. The promoter of GAP-43 is unusual in sequence and structure.
  • GAP-43 promoter The lack of a TATA box and consequent use of multiple start sites cause the GAP-43 promoter to resemble promoters of constitutively expressed housekeeping genes. However, the GAP-43 promoter lacks the consensus Sp-1 binding sites (GGGCGGG) that have been correlated with the promoters of housekeeping genes (Dynan, Trends Genet. 2:196-197 (1986)).
  • GAP-43 tightly regulated expression of GAP-43 in development, its specificity to neurons, and its inducibility in particular neurons in the adult sugest that it does not belong to this class of genes.
  • GAP-43 is regulated differently in the central and peripheral nervous systems. For example, axotomy of mammalian central neurons does not cause increased GAP-43 expression and transport, whereas axotomy of a peripheral nerve does (Skene et al, J. Cell Biol. 89:96-103 (1981). As described hereinabove, GAP-43 does not appear to be i ⁇ eversibly repressed in the CNS, and may play a role in plasticity other than in axonal growth (Benowitz et al. , T.I.N.S. 70:527-532 (1987)), but it is clear that there is a difference in regulation centrally and peripherally.
  • Thy-1 a gene expressed in, although not limited to, neurons, has been demonstrated to be expressed in a developmentally regulated, tissue-specific fashion at the transcriptional level, and also lacks a TATA box and Sp-1 binding sites (Spanopoulou et al., Molec. Cell. Biol 8:3847-3856 (1988)).
  • the choice of transcriptional start sites in the Thy-1 promoter can vary between expressing tissues, with upstream start sites being more prominent in the brain (Spanopoulou et al., Molec. Cell. Biol. 8:3847-3856 (1988)). This suggests an additional level of control in brain versus other tissues for both GAP-43 and Thy-1.
  • TATTCATG consensus Pit-1 binding site
  • GAP-43 promoter Another remarkable feature of the GAP-43 promoter is the presence of long homopurine-homopyrimidine stretches. These are interesting because they may bind proteins specific to GAGA stretches (Biggin et al, Cell 53:699-711 (1988); Gilmour et al., Sdence 245:1487-1490 (1989)), and because they have the potential to take on a triple stranded conformation called H-DNA. Such homopolymer regions have been found to be overrepresented in the 5' ends of eukaryotic and eukaryotic viral genes, leading to the speculation that they may somehow be involved in transcriptional control (Wells et al., FASEB J.
  • H-DNA could serve as a represser of transcription by directly blocking access to DNA in its immediate vicinity (Maher et al, Science 245:725-730 (1989)).
  • additional embodiments of the present invention comprise, inter alia, a nucleotide sequence as shown in Figure 13 encoding genomic GAP-43, or a functional or chemical derivative thereof, as well as a nucleotide sequence as shown in Figure 14 encoding the GAP-43 promoter, or a functional or chemical derivative thereof.
  • the GAP-43 promoter of the invention will be of great utility, not only in modifying the activity of GAP-43 itself, but as a means of achieving desired alterations -in expression of other stracmral genes, using methods well known to those -of skill.
  • this aspect of the invention is directed to a promoter substantially as shown in Figure 14, characterized in that it contains multiple start sites and a consensus Pit-1 binding site, but lacks a TATA box and consensus Sp-1 binding sites, and further characterized in that it comprises long homopurine-homopurimidine stretches capable of taking on triple stranded (H-DNA) conformation.
  • DNA expression vectors comprising the stracmral gene as described above, host cells transfected with these vectors, and the proteins produced thereby.
  • G-A Major Component of the Neuronal Growth Cone Membrane is the GTP Binding Protein.
  • the neuronal growth cone contains specialized transduction machinery which converts signals from the microenvironment into directed growth of axons or dendrites.
  • Subcellular fractions from neonatal rat brain that are enriched in growth cone membranes have simple and distinctive protein composition.
  • the two major non-cytoskeletal proteins in growth cone membrane preparations have molecular weights of 40,000 and 35,000. By electrophoretic, immunologic and partial protein sequence criteria, these proteins have been identified as the alpha and beta subunits of the GTP binding protein, G c .
  • Immunohistologic staining of neuronally differentiated rat pheochromocytoma cells demonstrates high concentrations of the alpha subunit of G 0 at the distal tips of cellular processes.
  • the distal tip of a neuronal axon has a unique ultrastructure termed the growth cone, which is thought to be critical for this process (Bray, et al, Ann. Rev. Cell Biol. 4:43 (1988)).
  • the membrane of the axonal growth cone can be fractionated from other neuronal constituents (Pfenninger, et al., Cell 35:573 (1983); Gordon-Weeks, etal, Neuros ⁇ ence 73:119 (1984); Ellis, et al., J. Cell Biol 707:1977 (1985)). It is composed of only a few major proteins, and several of these proteins have been identified: tubulin, actin, and the neural-specific, growth-related protein, GAP-43 (Pfenninger, etal., Cell 35:573 (1983); Ellis, et al., J. Cell Biol 101:19 ⁇ 7 (1985); Simkowitz, et al., J.
  • a growth cone membrane fraction was prepared from neonatal rat brain (Pfenninger, et al., Cell 35:573 (1983); Ellis, et al., J. Cell Biol 707:1977 (1985); Simkowitz, et al., J. Neurosd. 9:1004 (1989); Cheng, et al., J. Biol. Chem. 263:3935 (1988)).
  • This preparation has a simple protein composition by SDS-PAGE (Ellis, et al., J. Cell Biol. 707:1977 (1985); Simkowitz, et al., J. Neurosd. 9:1004 (1989); Cheng, et al., J. Biol. Chem. 263:3935 (1988)).
  • the most intensely stained ban, migrating at 50-55,000 daltons, has been identified as tubulin (Simkowitz, et al., J. Neurosd. 9:1004
  • p34 and p38 have similar molecular weights to the alpha and beta subunits of the GTP-binding protein, G 0 (Stryer, et al, Ann. Rev. Cell Biol. 2:391 (1986); Gilman, Ann. Rev. Biochem. 56:615 (1987)).
  • Co-electrophoresis of the growth cone membranes with purified bovine grain G 0 demonstrated that p34 co-migrates with the beta subunit, and p38 with the alpha subunit of Glois.
  • Immunoblotting demonstrated that p34 reacts with an anti-beta subunit antiseram, and that p38 reacts with an anti-alpha subunit G 0 antiseram.
  • the predominant protein species of p38 must be alpha,,, because equal protein concentrations of p38 and alpha ⁇ , as determined by Coomassie blue staining, exhibited identical immunoreactivity. The same was true for p34 and the beta subunit of G c . Alph ⁇ j subunit was about 10-fold less reactive than alpha,, with this antiseram (Gilman, Ann. Rev. Biochem.
  • peptides were from regions where beta, and betaa subunits are identical and the third contained a mixture of the sequences for beta, and betaj.
  • the alpha and beta subunits of G 0 are major constituents of the growth cone membrane subcellular fraction.
  • G olf a related G protein, G olf , is localized to the terminal region of primary olfactory neurons in the adult (Jones, et al., Sdence 244:790 (1989)), and that G 0 stains throughout cultured primary neurons but is concentrated at regions of cell-cell contact (Jones, et al., Sdence 244:790 (1989)).
  • protease inhibitors were employed throughout the procedure: 100 ug/ml soybean trypsin inhibitor, 1 ug/ml pepstatin A, 30 uM leupeptin, and 1 mM PMSF.
  • the crude brain homogenate was layered over a step gradient of sucrose at 0.75 M, 1.0 M and 2.2 M. The gradient was centrifuged at 250,000 x g for 40 min, and the 0.32/0.75 M interface was collected as the growth cone particle fraction. This fraction was lysed in 5 mM Tris-HCl, pH 7.6, and the membranes were collected by centrifugation at 250,000 x g for 40 min.
  • Bovine brain G 0 was prepared as described (Bray, et al., Ami. Rev. Cell Biol. 4:43 (1988)).
  • PC12 cells were grown on poly-D-lysine treated coverslips for 48 hours in the presence of 100 ng ml nerve growth factor.
  • the cells were fixed with 3.7% formaldehyde in phosphate buffered saline (PBS), and then permeabilized with 0.1 % Triton X-100. After incubation with 5 mg/ml bovine serum albumin in PBS, the cells were incubated with 1: 1000 anti-bovine brain alpha o antiseram for 1 hour at 23°C, rinsed with PBS, and incubated with 0.3% H 2 O 2 for 15 minutes to reduce background. Bound rabbit immuno- globulin was detected by use of the Texas red conjugated donkey anti-rabbit IgG (Jackson Immunologicals).
  • Immunoblotting revealed lpha ⁇ immunoreactivity migrating at the position of alpha Coomassie blue staining in both the purified G 0 preparation and the growth cone membrane preparation.
  • the gels were loaded such that Coomassie blue labeling of alpha ⁇ was identical to that of P38, and similarly matched for other pairs.
  • the Coomassie stained gels were ran in parallel. Note that the pairs also were immunostained to the same degree, as the total protein was increased, demonstrating that p38 is as immunoreactive as authentic alpha o with this antiseram.
  • the partial protein sequence for p34 and p38 is identical to that of G...
  • the partial protein sequence for p34 and p38 is shown in Figure 16.
  • the sequence of three peptides from p38 matches the sequence of three peptides from alpha, from rat brain (Goh, Sdence 244:980 (1989)).
  • the sequence of three peptides from p34 is compared to that of betaj and beta s subunits from bovine brain (Fong, et al, Proc. Natl Acad. Sd. USA 84:3792 (1987)). Note that two peptides are identical to regions in which beta, and betaj are identical.
  • the other peptide contains a mixture of the sequences for beta x and beta 2 .
  • G proteins in general, couple transmembrane receptors -to intracellular signalling systems, although the role of G 0 , which is expressed primarily in brain, has not been clear (Stryer, et al, Ann. Rev. Cell Biol 2:391 (1986); Gilman, Ann. Rev. Biochem. 56:615 (1987); Neer, Nature 333:129 (1988): Ross, Neuron 3:141 (1989)).
  • G Computing can interact with a number of cell surface receptors and may affect a variety of intracellular signalling systems including phospholipase C, phospholipase A 2 , potassium channels and calcium channels (Skene, et al., Sdence 233:783 (1986); Stryer, et al, Ann. Rev. Cell Biol 2:391 (1986); Neer, Nature 333:129 (1988); Ross, Neuron 3:141 (1989); Brown, et al., Am. J. Physiol 254.-H401 (1988)).
  • the strikingly high levels of G 0 in the growth cone membrane which are comparable to those of the retinal G protein, transducin, in rod and cone outer segments, strongly suggest a G c -based transduction system in growth cones.
  • G proteins have been proved crucial to developmental mo ⁇ hogenesis in the slime mold, Dictyostelium, where chemotaxis towards cAMP is transduced via a G protein (Snaar-Jagalska, et al, F.E.B.S. Lett. 232:148
  • signals from pathways or targets in the developing nervous system may bind to a G 0 -linked receptor, or receptors, in the growth cone membrane, and thereby alter the level of intracellular second messengers, and hence growth cone motility.
  • these signals, receptors, and second messengers are unknown at present.
  • One class of candidate receptors are the cell adhesion molecules, N-CAM and LI, which are localized to the neuronal growth cone (Letoumeau, et al., Development 105:505 (1989)).
  • Antibodies to thse molecules alter calcium levels and phosphotidylinositol metabolism in PC12 cells, and the effect of these antibodies is blocked by the G protein antagonist pertussis toxin (Van Hooff, et al., J. Cell Biol 108:1115 (1989)).
  • GAP-43 Another growth cone enriched molecule, GAP-43, also exists in discrete regions of the adult brain (Benowitz, et al, Trends Neurosd. 70:527 (1987); Skene, Ann. Rev. Neurosd. 12:127 (1989)).
  • the localization of GAP-43, the nature of its gene regulation, and especially the correlation of its phosphorylation state with long-term potentiation in the hippocampal slice (Routtenberg, N.7. Acad. Sci.
  • GAP-43 in synaptic plasticity in the adult (Benowitz, et al, Trends Neurosd. 10:527 (1987); Skene, H.J.P., Ann. Rev. Neurosd. 72:127 (1989)). It is noteworthy that pertussis toxin blocks long-term potentiation, perhaps implicating G 0 in this process as well (Goh, Sdence 244:980 (1989)). Hence, G 0 may transduce regulatory signals for axonal extension during neuronal development and for synaptic plasticity in the adult nervous system.
  • the nerve growth cone controls neuronal form by sensing signals from neighboring cells, matrix and targets, and by transducing this information into structural changes.
  • the consequent directed growth directs establishment of proper connections during development.
  • Adult CNS neurons have relatively unimpressive regenerative capacity, which is a vexing clinical problem.
  • inhibitors may be critical in explaining poor regeneration within the adiilt
  • G c The most prominent non-cytoskeletal protein associated with the growth cone membrane is G c , a member of the heterotrimetric G protein family.
  • GAP-43 The great abundance of G 0 in the growth cone, and its regulation by the growth associated protein, GAP-43, suggest that G 0 might be a key intermediary in the transduction of mo ⁇ hogenetic information. This model predicts that G protein activation or inhibition should affect neuronal growth.
  • the following experiments demonstrate that G proteins act as mediators of neuronal growth in both CNS and PNS cells.
  • Embryonic chick sympathetic neurons and retinal ganglion cells were isolated as follows. Sympathetic chains, isolated from the thoraco-lumbar regions of day eight to twelve chick embryos or retina dissected from day 7 chick embryos, were incubated for 15 minutes in 0.25% trypsin and 0.5 % collagenase in HBSS at 37°C. The tissue was then triturated in growth medium (F12 with 10% fetal bovine serum, glutamine, penicillin/streptomycin, and, for sympathetic neurons, 5 ng of NGF/ml), 15 times with a fire polished pasture pipet.
  • growth medium F12 with 10% fetal bovine serum, glutamine, penicillin/streptomycin, and, for sympathetic neurons, 5 ng of NGF/ml
  • Cells were diluted to a density of 1 x 10 4 cells/ml, preplated on an untreated tissue culture substrate for 3 hours to allow fibroblasts to attach, and then plated onto glass chamber slides coated with either poly-Lrlysine (5 mg/cm 2 for 30 minutes at 20 °C) or laminin (4 mg/cm 2 for 60 minutes at 20 °C).
  • Poly-Lrlysine 5 mg/cm 2 for 30 minutes at 20 °C
  • laminin 4 mg/cm 2 for 60 minutes at 20 °C.
  • Conditions for inhibiting or stimulating G proteins Pertussis toxin treatment was performed by incubating neurons in floating culture with either 200 ng/ml of freshly prepared toxin (Cal Biochem), or PBS alone as control for 3 hours and then plating cells in parallel.
  • Aluminum flouride (A1F) or mastoparan were added to cultures 45 minutes after plating.
  • Conditions for electroporation of primary neurons were developed to facilitate entry of relatively small molecules (MW ⁇ 1000) into live cells.
  • sulfo- rhodamine a highly ionic rhodamine analog which does not penetrate intact cells
  • conditions were optimized to maintain > 80% cell viability and achieve > 95% cell permeability.
  • the integrity of the plasmalemma was only temporarily violated as sulfo-rhodamine could be detected within neurons when it was present during the electroporation, but not when it was added as little as 1 minute post- electroporation.
  • Electroporations were performed in growth medium at 500 V and 1 microFarad in the presence of 1 mM GTP7S, 1 mM GDP beta-S or PBS alone. Fifteen discharges were each spaced by 5 seconds and then cells were plated. To assess the efficiency of electroporation, cells were incubated with or without electroporation in the presence of sulfo-rhodamine, plated, allowed to attach, washed with growth medium and fixed after 18 hours in culture.
  • pertussis toxin inhibits receptor mediated activation of G 0 and Gj via irreversible ADP-ribosylation. Treatment with pertussis toxin causes a significant increase in both the total number of neurites per cell and the total length of neurites per cell. A1F acts as an activator of G-proteins and has been shown in other cell types to stimulate G-protein mediated events.
  • Mastoparan a wasp venom toxin, stimulates G 0 and G ; through a reversible interaction with their receptor binding domain. Both aluminum fluoride and mastoparan dramatically reduced process formation. Although prolonged exposure to either agent was clearly toxic, the inhibitory effect on neurite outgrowth was reversible. Cell death in the mastoparan and A1F treated cultures was approximately 60%.
  • the non-hydrolyzable guanine nucleotide analogs GTP ⁇ S and GDP/3S bind to and maintain G proteins in the active and inactive states, respectively. Electroporation of GTP7S, GDP/3S or control buffer (PBS) was performed as described in cultures of embryonic chick sympathetic neurons. Neurons were plated immediately after electroporation, fixed one hour post-plating and examined by phase-contrast or Normarski optics.
  • G-protein inhibition caused an increase in the total number of neurites per cell, the total length of neurites per cell, and the average neurite length in cells. In contrast, neurite extension was significantly diminished in cells receiving GTP7S.
  • Retinal ganglion cells exhibit similar increases in neurite extension when treated with pertussis toxin or with electroporation of GDP/Ss. GTP7S electroporation reduces neurite outgrowth, but to a lesser degree than in sympathetic neurons.
  • Neuronal plasticity depends upon a complex interaction involving such factors as G proteins and inhibitory or stimulatory ligands.
  • G protein modulation upon neuronal plasticity, depends on the particular environment and the balance between inhibitory and stimulatory ligands.
  • G proteins There are many cell-specific receptors that guide growth, some of which are known to be coupled via G proteins.
  • serotonin application to Heliosome neurons causes growth cone collapse and cessation of axon elongation (Haydon et al., Sdence 226:561-564 (1984)), which is compatible with the observations reported here.
  • pertussis toxin has been shown to modify LTP, a process now believed to involve physical changes in nerve terminal size. Given the abundance of G 0 in growth cones, and the effect upon neurons from both the PNS and CNS, it appears likely that G proteins may be relatively universal mediators of growth cone responsiveness.
  • G proteins may be to mediate inhibitory, or "stop" signals. Such signals are important in neuronal development and plasticity. Oligodendrocyte proteins have been isolated of 35 K and 250 K MW which inhibit growth of central and peripheral nervous system neurons. Although each of these systems may bear specific receptors for negative signals, perhaps they share the G protein transduction machinery. This is of clinical significance, since it is important to devise means to encourage regeneration of CNS neurons from many sites, and inhibition of G proteins may provide such tools.
  • GAP-43 stimulates GTP7S binding, GTPase activity and GDP release in the same fashion as do transmembrane G-linked receptors.
  • GAP- 43 differs from receptors in that it acts on the isolated alphag subunit just as effectively as on the heterotrimeric G c .
  • Pertussis toxin blocks receptor/G 0 interaction, but not GAP-43/G 0 interaction.
  • the relative ability of fifteen synthetic peptides to stimulate G 0 demonstrates that the first 25 amino acids of GAP-43 are important for its effect on G 0 . It has been shown that the major form of GAP-43 isolated from brain is phosphorylated by protein kinase C at position 41.
  • GAP-43 Dephosphorylation of GAP-43 does not alter its potency for stimulating G 0 .
  • Calmodulin can bind to GAP-43, but 20 uM calmodulin does not alter the GAP-43 effect on Genfin. Ghilst may integrate extracellular and intraneuronal growth clues in the growth cone.
  • GAP-43 could be an upstream regulator of G 0 activity or a downstream effector of activated G 0 .
  • Such inhibitory activities are components of brain membranes, myelin, and somites (Cox et al., Neuron 4:31-37 (1990); Raper et al, Neuron 4:21-29 (1990); Schwab, Trends Neurosd. 73:452-456 (1990); (Davies et al., Bioessays 13: 11-15 (1991); Davies et al, Neuron 4: 11-20 (1990)).
  • Their role has been speculated to be critical to the prevention of regrowth of injured axons in the CNS, to pathway guidance through somites, and to the recognition of targets (Aveldano et al, J. Neurochem. 57:250-257 (1991)).
  • Kaphammer et al found that collapse of growth cones can be induced by brain membranes, an effect exerted both upon CNS and PNS neurons (Kapfhammer et al., J. Neurosd. 7:201-212 (1987). Others, using growth in cell culture chamber systems, have partially isolated myelin-associated proteins which inhibit growth of neurons (Schwab, 1990). The transduction mechanisms which are responsible for registering inhibitory signals and triggering growth cone collapse are largely unexplored. It is conceivable that inhibitory mechanisms might be shared between neurons of different types, although the specific signals and their receptors might differ.
  • G 0 is the most prominent non-cytoskeletal protein of the growth cone membrane, which suggests that G 0 may be integral in the response to environmental signals that direct pathway and target selection.
  • the inventors have examined the role of G proteins in growth cone collapse and found that collapse stimulated by both brain membranes and myelin has a large component dependent upon pertussis toxin-sensitive G proteins.
  • the membranes were prepared from E10 chick brains as described previously. (Raper et al., Neuron 4:21-29 (1990)). The crude collapsing activity was obtained from detergent-solubilized membrane extracts as described by Raper and Kapfhammer. (Raper et al., Neuron 4:21-29 (1990)).
  • the membranes were suspended in 2% CHAPS/PBS and the extract was centrifuged (100,000 g for 1 hour). The supernatant was dialyzed against PBS and then F12 medium to remove the detergent. The dialysate was used as the crude growth cone collapsing activity.
  • Each DRG was removed from E7 chick embryos and explanted onto laminin-coated glass chamber slides in
  • F12 medium including NGF, fetal calf serum, streptomycin, and penicillin.
  • CNS myelin was prepared from adult rat cerebral cortex (Norton etal., J. Neurochem. 27:749-757 (1973)), and myelin proteins were solubilized by
  • Brain membranes isolated by standard techniques (Raper et al., Neuron 4:21-29 (1990)), increase the percentage of collapsed growth cones, and do so in a dose-dependent way, with maximal effects exerted by about 1.2 mg/ml of brain extract, as shown in Figure 18.
  • Preincubation of the dorsal root ganglion explants with pertussis toxin markedly attenuates this collapse.
  • Preincubation with 100 ng/ml shifts the dose response curve by six-fold and with 200 ng/ml by ten-fold. This suggests that the collapsing activity of the brain extracts exerts a significant proportion of its effect via pertussis toxin- sensitive G proteins. Additional mechanisms that are non-pertussis toxin- sensitive may be brought into play at the highest concentrations of brain extract.
  • Brain extracts have been shown also to cause collapse of retinal neurons from chick, a finding of clinical relevance in terms of the regrowth of an inured optic nerve (David et al, Sdence 74:931-933 (1981)).
  • FIG 20 shows that about 26% of growth cones from the explanted retina are collapsed under basal conditions.
  • retinal growth cones also collapse with exposure to brain extract, as shown in Figure 20.
  • this collapse is markedly attenuated by pretreatment with pertussis toxin, suggesting that central nervous system as well as peripheral nervous system growth cone collapse is mediated in large part by pertussis toxin-sensitive G proteins.
  • CNS myelin protein solubilized by octyl glucoside, causes growth cone collapse of the dorsal root ganglia.
  • the addition of the myelin extract to the dorsal root ganglion cultures causes an increase in collapsed growth cones from 26% to 68%.
  • Pretreatment of the dorsal root ganglia with pertussis toxin reduces the percentage of growth cones which collapse with exposure to myelin from 68% to 38%.
  • G proteins facilitate the growth cone collapse stimulated by myelin and brain membranes. Therefore, one embodiment of the invention relates to the use of agents which regulate the action of G proteins, to modulate the inhibitory action on neuronal growth executed by components of myelin and brain membranes.
  • GAP-43 would be used to regulate G c activity, thereby modulating the inhibitory action of myelin and brain membranes.
  • GAP-43 is a Novel Internal Regulator of Protein Binding
  • the present invention is directed to the su ⁇ rising discovery that GAP-43 acts within the cell to modify the binding capacity of other cell proteins, including that of G 0 .
  • GAP-43 acts within the cell to modify the binding capacity of other cell proteins, including that of G 0 .
  • IRP internal regulatory proteins
  • synthetic peptides comprising the amino terminus amino acids of GAP-43 duplicate exactly the modulation in GTP binding by G c that is caused by the intact GAP-43 protein.
  • a peptide comprising the first 24 amino acids of GAP-43 stimulates GTP7S binding to the same level as GAP-43, acting as a full GAP-
  • a related embodiment of the invention is directed to nucleotide sequences encoding the synthetic IRP peptides described above, which sequences will easily be determined by those of skill who have appreciated the teachings of the present invention.
  • a further aspect of the invention is directed to the discovery that a consensus amino acid sequence is found in GAP-43 and beta adrenergic receptors, said sequence comprising
  • cysteines of the IRPs and IRP peptides of the invention may be prone to palmitoylation.
  • a method of stimulating the binding activity of a desired protein comprising introducing into an environment comprising said desired protein and its binding substrate an effective amount of an IRP peptide.
  • the desired protein is preferably a G protein, and, most preferably, G 0 .
  • the preferred binding substrate is GTP, and GTP7S is most preferred.
  • the environment is preferably that inside a living cell, which may be a central or peripheral neural cell.
  • compositions and methods of the invention are directed, inter alia, to mechanisms involved in neuronal growth and synaptic plasticity. It may be desirable, for a given medicinal indication, to reduce, as well as enhance, neural growth or plasticity. This may be accomplished, for example, by administering antibodies directed against the IRP peptides of the invention, or against the sites at which such IRP peptides have their physiological effect.
  • the invention is directed to antibodies, preferably monoclonal antibodies, directed against the IRP peptides of the invention, and to functional or chemical derivatives thereof, said antibodies or their said derivatives being optionally detectably or therapeutically labeled.
  • the invention is directed to pharmaceutical compositions comprising the IRP peptide of the invention, together with a pharmaceutically acceptable carrier, and optionally comprising one or more therapeutically effective agents, as well as to pharmaceutical compositions comprising an antibody directed against the IRP peptides of the invention, together with a pharmaceutically acceptable carrier, and optionally comprising one or more therapeutically effective agents.
  • pharmaceutical compositions of the invention will be accomplished by those of skill wihtout undue experimentation, keeping in mind those principles of administration as set forth herein and as are well known in the art.
  • the invention is directed to a method of modulating structural remodeling in a neural cell, comprising administering to said cell an effective amount of the compositions of the invention.
  • the GAP-43 sequences of the invention have been used to isolate a G-liko protein from neural cells.
  • a protein of MW 39,000 has been found to bind specifically a GAP-43 with high affinity.
  • Cell extracts were introduced into columns containing GAP-43 in a buffer comprising 50 mM Tris, 1 mM CaCl 2 , and 1 mM MgCl 2 .
  • the protein elutes in a single band witii equimolar EDTA buffer.
  • the protein does not react with polyclonal antibody to G protein. It is thus a distinct and novel protein associated with growing neurons, and forms an additional embodiment of the invention.
  • IRP peptides according to the invention may be produced by any known means, for example, using recombinant genetic methods as described hereinabove, and that nucleotide sequences encoding the IRP peptides of the invention may be deduced and optomized for a desired host expression system with the exercise of merely routine skill.
  • the significance of the novel compositions and methods of the invention in modifying cellular transduction systems such as G ⁇ is enhanced when considered in conjunction with the demonstration herein that G 0 is a major noncytoskeletal protein present in neuronal growth cones.
  • GAP-43 the function of which has not previously been known, modulates G ⁇ activity, is evidence that GAP-43 is a long sought "missing link" between first and second messengers in cellular transduction systems.
  • GAP-43 may interact with G 0 in a similar place in the molecule as do the cytosolic domains of the G protein- linked receptors.
  • IRP peptides may be, for example, stimulatory or inhibitory.
  • the IRPs and IRP peptides, or other substances having such internal regulatory action may have any of the possible pharmacologic effects, such as are known to those of skill in the art and are described in standard reference works including, for example, Remington 's Pharmaceutical Sdences, 16th ed., Mack Publishing Co., Easton, Penn. (1980), and Goodman et al, The Pharmacological Basis of Therapeutics, 7th ed. , Macmillan Publishing Co., New York (1985), or the current editions thereof.
  • a method of screening for a substance capable of modulating the binding activity of a desired protein comprising introducing into an environment comprising said desired protein and its binding substrate a substance which it is desired to screen, and measuring the increase or decrease in substrate binding relative to substrate binding in the absence of said substance.
  • the desired protein may be a G protein, and, preferably, is G 0 .
  • GAP-43 Stimulates GTP'yS Binding to G..
  • GAP-43 for G ⁇ measured in the assays described herein is consistent with in vivo conditions. All assays were conducted in the presence of 200 ug/ml BSA, so a nonspecific protein effect by GAP-43 cannot explain the stimulation of GTP7S binding. G c is known to be partially inactivated during preincubation at 30°C without GTP7S (O'Dowd, et al., J. Biol. Chem. 263: 15985-15992 (1988)). GAP-43 does not affect the degree of G 0 thermal instability.
  • the other group of proteins known to stimulate GTP7S binding to G proteins are hormone and neurotransmitter receptors.
  • Overall hormone and neurotransmitter receptor structure e.g. , a large extracellular region and seven transmembrane domains
  • the inventors thus searched for homologies between these proteins, focusing on those domains thought to interact with G proteins.
  • site-directed mutagenesis and peptide competition studies have implicated the carboxyl end of the third cytoplasmic loop and the proximal end of the cytoplasmic tail in G-protein coupling (Konig, et al, Proc. Natl. Acad. Sd. U.S ⁇ .
  • B2-adrenergic receptor linkage with G is interrupted most 5 specifically by a point mutation at a palmitylated cysteine in the cytoplasmic tail of the protein (O'Dowd, et al., J. Biol. Chem. 264:7564-7569 (1989)).
  • cysteines in the amino terminus of GAP-43 which are required for G 0 regulation are also subject to palmitoylation in vivo (Skene, et al., J. Cell Biol. 108:613-624 (1989)).
  • the amino terminal portion of the cytoplasmic tail from a series of receptors reveals a consensus sequence of hydrophobic-leu-cys-cys-x-basic-basic (Fig. 22).
  • the cysteines where studied, undergo palmitoylation (Skene, et al., J. Cell Biol. 108:613-624 (1989); O'Dowd, et al., J. Biol. Chem. 264:7564-7569 (1989); Ovchinnikov, et al., FEBS Letts.
  • GTP7S binding to G proteins are unknown.
  • GAP-43 is not homologous to this peptide.
  • GAP-43 has previously been shown to bind calmodulin, an interaction which is enhanced in the absence of calcium (Alexander, et al., J. Biol. Chem. 262:6108-6113 (1987)). The physiologic relevance of this association is unclear.
  • the addition of low concentrations of Ca+ +-calmodulin or EGTA-calmodulin to G c causes no change in the ability of GAP-43 to stimulate (35S)GTP7S binding.
  • higher concentrations of calmodulin lead to dose-dependent reduction in GAP-43-stimulated (35S)GTP7S binding ( Figure 23).
  • the present data provide a growth cone mechanism for the coordination of extracellular signals with the expression of intracellular growth associated proteins during neuronal mo ⁇ hogenesis.
  • the strikingly high levels of the alpha and beta subunits of G 0 in the growth cone membrane suggest a major role for G c in neurite regulation.
  • the G 0 concentration in the growth cone membrane exceeds that of another G protein, transducin, in the highly specialized outer segment of retinal photoreceptor cells.
  • transducin in the highly specialized outer segment of retinal photoreceptor cells.
  • it is a link between the binding of extracellular signals to transmembrane receptors and the regulation of enzymes or ion channels which modulate intracellular second messengers.
  • the alpha polypeptide exists in a GDP-bound state until an agonist-receptor complex causes the exchange of GTP for GDP.
  • the GTP-bound activated alpha subunit then exerts its action on second messenger systems.
  • Endogenous alpha subunit GTPase activity terminates signal transduction.
  • G 0 is predominantly expressed in brain, where it is the major form of G protein. In adults, it is found in the neuropil (-), and the present data localize G c to the tips of neurites in growing cells where it is the major non- cytoskeletal protein.
  • G c may respond to a variety of receptors and in turn regulate a number of intracellular systems, including calcium channels, potassium channels, phospholipase G and phospholipase A 2 .
  • GAP-43 The restricted localization of G c suggests that the protein's regulation or action is mediated by one or more neuronal-specific molecules.
  • GAP-43 is expressed only in neurons, and the protein is enriched in the growth cone. Therefore, the present inventors knew whether GAP-43 might interact with G 0 .
  • GAP-43 enhances GTP7S binding to G c .
  • a small region of GAP-43 defined by a synthetic decapeptide, exerts this action. Stimulation of GTP7S binding by GAP-43 is similar to that by agonist-receptor complexes, and the decapeptide sequence has homology with these receptors.
  • GAP-43 mimics transmembrane receptors and activates G 0 , creating a GTP-bound alpha subunit which then triggers an intracellular second messenger system.
  • GAP-43 binding to G 0 might function primarily to disrupt G 0 -receptor or G 0 -effector interactions.
  • GAP-43 is an effector of G 0 activation by receptor in some as yet unknown manner.
  • the modulation of a G 0 cone transduction system by a growth associated protein, GAP-43 provides a mechanism to integrate extracellular signals with an intracellular program for neuronal growth.
  • GAP-43 might synergistically enhance the response of G 0 to extracellular ligands, or decrease responsiveness to ligands by overridin the dependency of G 0 on receptor. In the later case, removal of GAP-43, as occurs during synapse formation, would restore sensitivity to extracellular ligands.
  • the net effect of GAP-43 action on receptor effectiveness would depend on the relative concentrations of the components.
  • GAP-43 is unique among G-protein regulators in that it is an intracellular protein with no presently known capacity to respond directly to extracellular ligands. However, the intracellular regulation of membrane bound GTPase proteins does have precedence. Normal RAS proteins are stimulated by a widely distributed 120 kD intracellular protein, GAP, Despite the similarity in their names, GAP and GAP-43 are unrelated proteins.
  • the cysteines in the region of GAP-43 and receptors which stimulate GTP7S binding to G 0 are subject to palmitoylation.
  • the amino terminal peptides and probably the GAP-43 exist in a non-palmitylated state.
  • the relative ability of palmitylated versus non-palmitylated GAP-43, and G-linked receptors, to stimulate G proteins is unknown. It is possible that rapid palmitoylation-depalmitoylation plays a regulatory role for these proteins.
  • the persistence of G c expression in the adult nervous system (Worley, et al., Proc. Natl Acad. Sci.
  • GJG ⁇ antagonist pertussis toxin, blocks long-term potentiation, perhaps implicating G 0 in this process as well (Goh, et al., Sdence 244:980 (1989)).
  • G 0 may transduce intracellular and extracellular signals for neurite extension during development and for synaptic plasticity in the adult nervous system.
  • GAP-43 enhances filopodial formation in non-neuronal cells
  • filopodia To determine whether the production of filopodia is dependent on contact with a substratum, cells were fixed in suspension, and filopodia quantitated. More filopodia-bearing cells are seen among the GAP-43 expressing cells under these conditions (shown as zero time point in Figure 24). The presence of filopodia is not significantly altered by varying the length of time between trypsinization and analysis from 5 minutes to 120 minutes. Thus, GAP-43 is not merely altering the recovery from trypsinization.
  • the GAP-43 plasmids were all derived from CDM-8 and contain an SV40 origin of replication, and the rat GAP-43 cDNA sequence under the control of a CMV promoter.
  • the point mutations of arg 6 to gly, of arg 7 to gly and of lys 9 to gly were created from the GAP-43 vector by oligonucleotide- directed mutagenesis, as described previously for the cys 3,4 to thr substitutions.
  • the CAT and GAP-43/CAT expression vectors were constructed as described previously.
  • the GAP-43(1-10)/CAT constructs encode a stretch of 17 amino acid residues, VDLQASLARFSGAKEAK, linking GAP-43 to CAT.
  • the GAP-43(l-40)/CAT and GAP-43(1-6)CAT proteins are linked by ARVDLQASLARFSGAKEAK. All mutations and fusions were confirmed by DNA sequencing. Non-neuronal cell culm re and transfection
  • A431 epitiielial cells were maintained in DMEM, 7.5% fetal bovine serum. DNA transfections were by the calcium phosphate procedure, and included a neomycin resistance gene expression vector, pDOJ (Bloch et al., Mol. Cell Biol 9:5434-5439(1989)), and a 5-fold excess of a GAP-43 expression vector (Zuber et al., Sdence 244:1193-1195 (1989a)). Stable transfectants were selected in 400 ⁇ g/ml G418 and then screened for protein expression by immunoblotting. After selection, clones were maintained without G418. Those clones with the highest levels of expression were used in subsequent experiments.
  • COS-7 cells were transfected with equal amounts of different DNAs by the DEAE-dextran method or by electroporation. Forty hours after transfection, cells were analyzed in the filopodial and spreading assays.
  • Filopodial formation was assessed by modification of previous methods. Cultures of CHO, or COS-7 cells were trypsinized at 30-50% confluency with 0.25% trypsin for 5 minutes at 37°C. The cells were then diluted into at lest 20 volumes of serum-containing medium and plated onto glass slides precoated with poly-Hysine at 50 mg/ml in PBS for 2 hour. After various incubation times at 37 °C (8 minutes in routine assays) the medium was aspirated and the cells fixed. CHO cells were fixed with 2% Glutaraldehyde in PBS, and stained with 0.1% Coomassie Blue in 25% isopropanol, 10% acetic acid.
  • COS-7 cells were fixed with 3.7% formaldehyde in PBS and then incubated for 1 hour in 5% normal goat serum, 0.1 % Tyriton X-100, PBS with 1:1000 rabbit anti-GAP-43 serum, or with 1 ⁇ g/ml anti-CAT antibody (5 '-3', Inc.). Bound antibody was detected by fluorescence after incubating with fluorescein- labelled goat anti-rabbit IgG. CHO cells with filopodia were quantitated by observation at 400X magnification with Nomarski optics. COS cells expressing the protein of interest were identified by fluorescence microscopy and then positive cells were scored positive or negative for any spike-like protrusion from the cell body greater than 2 ⁇ in length. In each experiment, 50-200 cells were counted. Each cell line or transient expression assay was examined in at least three separate experiments.
  • Filopodia are transient, so that few GAP-43 expressing COS or CHO cells exhibit them after 30 minutes. However, over the next 1 to 4 hours, the shape of GAP-43 cells can be distinguished from control cells by another attribute, the rate of cell spreading.
  • control COS cells are plated on poly-Hysine coated glass, cell spreading occurs over the first 60 minutes ( Figure 25). The rate of spreading by GAP-43-expressing cells is slower, although the degree of spreading eventually reaches the same level.
  • One day after plating cell shape is indistinguishable between control and GAP-43 cells. To examine the generality of these changes in cell spreading, stably transfected clones derived from CHO and A431 cells were also studied.
  • Control A431 cells spread more extensively than CHO or COS cells and therefore provide the most convenient assay system for cell spreading.
  • GAP- 43 expressing A431 clones spread less rapidly than do control lines, and a clear diminution in cell area is apparent 2 hours after plating on laminin-coated glass (Figure 25).
  • CHO clones expressing GAP-43 also exhibit a decrease in spreading, but since spreading is less extensive in control CHO cells, this is more difficult to quantitate.
  • Immunoblot analysis of GAP-43-expressing A431 cells demonstrates a GAP-43 concentration that is 10-50% of that found in whole neonatal rat brain, so that these effects on non-neuronal cell shape occur at physiologic levels of GAP-43 ( Figure 25).
  • GAP-43 The amino terminus of GAP-43 is necessary and sufficient to induce filopodia and to inhibit cell spreading
  • the ability of GAP-43 to alter non-neuronal cell shape provides a bioassay for determining its functional domains. This is useful since there is no simple way to test the action of such mutants on neuronal growth cones directly, due to the high levels of endogenous GAP-43 in all studied neuronal culture systems. As several potentially functional domains have been described in GAP-
  • GAP-43 action a chimeric protein containing an N-terminal fragment of GAP-
  • GAP-43 fused to the N-terminus of chloramphenicol acetyl transferase (CAT), was expressed in COS cells. This fusion protein caused the same increase in filopodia and decrease in spreading as does intact GAP-43. CAT is inactive in these assays. A series of GAP-43.CAT fusion proteins were examined to better define the active domain. The GAP-43(1-10)/CAT protein was fully active, but he GAP-43(l-6)/CAT protein caused no change in filopodia or spreading when compared to control cells expressing CAT.
  • CAT chloramphenicol acetyl transferase
  • the internal deletion protein and the GAP-43(l-40)/CAT fusion protein are capable of inducing filopodia in stably-transfected clones of CHO cells as effectively as in transiently-transfected COS cells ( Figure 26).
  • Basic residues are 6 and lvs 9 are required for cell shape modulation
  • G protein activator domain of receptors (Okamoto et al.. Cell 62:709-717 (1990a)), the inventors tested the importance of these residues for GAP-43 action.
  • Cysteine residues cvs 3 and cvs 4 are also required for GAP-43 effect on cell mo ⁇ hologv In addition to the cluster of basic residues, the amino terminus of GAP-
  • cysteine 43 contains two adjacent cysteine residues at position 3 and 4. These cysteines undergo reversible cycles of palmitoylation (Skene et al., J. Cell. Biol 108:613-624 (1989)) and this is almost certainly required for the membrane localization of the protein. It has previously been shown that substimtion of threonine for one or both cysteines shifts GAP-43 from membrane to cytosolic fractions. In addition, these cysteines are important for G protein activation by GAP-43. Palmitoylation of these residues reversibly blocks interaction of GAP-43 and G 0 , and a GAP-43(1-10) peptide with threonines substituted for cysteines does not stimulate G c . Thus, substitution of threonines for cys 3 and cys 4 in GAP-43 disrupts both G protein activation and membrane binding.
  • GAP-43(l-6)/CAT The membrane localization of three inactive proteins, GAP-43(l-6)/CAT, arg 6 - substituted GAP-43 and lys 9 -substituted GAP-43, demonstrates tiiat the mo ⁇ horegulatory activity is not conferred simply by those amino acid residues which cause membrane binding.
  • the G protein activator region of GAP-43 is required to alter cell shape
  • GAP-43 on cell shape is due to changes in G protein activity, the structural determinants should be very similar in the two assays.
  • the inventors also investigated the contributions of the cysteine residues at position 3 and 4 to GAP-43 stimulation of G ⁇ (Figure 35).
  • the effect of threonine substitution varies with the length of the peptide in which the substitution is made.
  • One or both cysteines can be replaced in the 1-25 peptide without loss of activity, but not in the 1-10 peptide where the dithreonine peptide is inactive ( Figure 35).
  • the cysteines may have a weak contribution to the active domain which can be compensated for by the secondary structure present in the larger peptides.
  • cysteines The function of these cysteines was also studied by modifying their side-chains in both GAP-43 protein and peptide. It had been previously shown that large hydrophobic modifications of the cysteines with palmitate renders the 1-25 peptide and the protein inactive. These studies showed that small, neutral reagents such as iodoacetamide and N-ethylmaleimide do not alter GAP-43 protein activity, even though they remove the free sulfhydryls at position 3 and 4 as shown in ( Figure 35). This supports the conclusions which resulted from the threonine-substituted 1-25 peptide. Thus, the basic residues in the 6-9 region appear to be the core region of the G protein activating domain in GAP-43.
  • the mutational analysis localizes the active portion of GAP-43 to the first ten amino acid residues. Remarkably, this stretch alone can confer GAP-
  • G protein-coupled receptors of the seven-transmembrane domain family and on the IGF-II receptor have defined a G protein activator sequence consisting of a basic-basic-x-basic (BBxB) or BBxxB motif (Okamoto et al., etal.. Cell 62:709-717 (1990); Okamoto et al., Cell 67:723-730 (1991), Figure 35).
  • This importance of this BBxB motif is consistent with receptor mutagenesis smdies (Ross, Neuron 3:141-152 (1989)) and is related to information on the stimulation of G proteins by the cationic amphiphilic peptide, mastoparan (Higashijima et al, J. Biol Chem. 265:14176-14186 (1990)).
  • GAP-43 has such a BBxB sequence at position 6-9, the N-terminal region which activates G 0 and modulates cell shape.
  • the first and third basic residues are critical for G, stimulation (Okamoto et al, Cell 62:709-717 (1990)), whereas the second is not (Okamoto et al, Biochem. Biophys. Res. Co m. 779:10-16 (1991b)).
  • the protein loses activity in the cell shape assays, whereas the second basic residue is not essential.
  • the G protein activating function of the IGF-II and 32-adrenergic receptors also depends on basic amino acids N-terminal to the BBxB or BBxxB sequence (Okamoto et al, Cell 62:709-717 (1990); Okamoto et al, Cell 67:723-730 (1991a)). No such basic residues are present N-terminal to this region in GAP-43, but he ⁇ -amino group of the protein is just five residues away and may contribute a positive charge.
  • the high degree of correspondence between the sequence required for G protein activation in receptors and that required for GAP-43 to modify non-neuronal cell shape strengthens the hypothesis that GAP-43 acts by modifying G protein transduction.
  • GAP-43 activation of G c can be distinguished from receptor stimulation of G proteins by several criteria, including pertussis toxin sensitivity, 07 subunit dependence and phospholipid requirements (Strittmatter et al., J. Biol Chem. 266:22465-22471 (1991)). There are also some differences in the sequences of the G protein activator region of receptors and GAP-43. For
  • GAP-43 the cysteine residues at position 3 and 4 appear to be involved in G 0 activation in addition to the basic residues. In the cell shape assays, GAP-43 is inactive without these cysteines. It may be that this dependence reflects a necessity for membrane association, which is also mediated by these cysteines, or that it reflects direct interruption of G protein interactions, or both.
  • GAP-43 accounts for its modulation of non-neuronal cell shape. This provides further evidence that GAP-43 regulates G protein transduction within cells, and supports the hypothesis that GAP-43 alters neuronal growth cone motility by such a mechanism. GAP-43 alters the mo ⁇ hologv of non-neuronal cells. The study of GAP-43 gene expression and protein localization have led to the hypothesis that it might influence growth cone motility. Thus, assessment of non-neuronal cell mo ⁇ hology was conducted in order to develop a functional bioassay for examining the molecular mechanism of GAP-
  • the membrane targeting domain of GAP-43 is not identical to its mo ⁇ horegulatory domain.
  • Three proteins, the GAP-43(1- 6)/CAT fusion, the arg 6 substituted GAP-43 and the lys 9 substituted GAP-43, are localized to the membrane but are inactive in the filopodial and spreading assays.
  • the membrane targeting region is not sufficient, by itself, to effect cell shape changes. This region may be necessary, but this is unclear at present, because all of the cysteine mutations which prevent membrane binding also alter G protein activator function. It is interesting to note that if membrane binding by the GAP-43(l-6)/CAT protein is dependent on palmitoylation, then a very short sequence is capable of directing palmitoylation.
  • Serine which is subject to phosphorylation by protein kinase C, and the calmodulin binding region of GAP-43 are not required for its action on non- neuronal cell shape. These sites may regulate activities not tested in this assay, or they might modulate the activity of the amino terminal domain of GAP-43, increasing or decreasing the concentration of GAP-43 required for cell shape modulation. Such a quantitative change might be too subtle to be readily detected in these assays.
  • GAP-43 accounts for its action on non-neuronal cell shape. GAP-43 stimulates both Gj and G 0 ; thus even though there is little or no G c in these non-neuronal cells, there is at least one potential G protein target for GAP-43 action.
  • G protein activity modulates non-neuronal cell shape.
  • the spreading of macrophages has been linked to the activation of G protein-coupled receptors and their second messengers (Petty et al., J. Cell Physiol 138:247-256 (1989)).
  • F-met-leu-phe peptides modify chemotaxis and actin polymerization via G proteins (Bengtsson et al, Proc. Natl. Acad. Sd. USA 87:2921-2925 (1990)).
  • G protein activity determines the degree of focus formation among clusters of fibroblasts (Klebe et al., Proc. Natl. Acad. Sd. USA 88:9588-9592 (1991)).
  • G ⁇ subunits are responsible for mutation in Dictyostelium (Deverotes, Sdence 245:1054-1058 (1989)), both of which disrupt developmental mo ⁇ hogenetic events.
  • Second messengers known to be dependent on G protein activation have well-characterized actions on actin cytoskeletal dynamics (Hartwig etal, Curr. Opin. Cell Biol. 3:87-97 (1991)).
  • G would alter growth cone motility.
  • G proteins respond to complexes between extracellular ligands and transmembrane receptors (Gilman, Annu. Rev. Biochem. 56:615-
  • G protein regulation by intracellular proteins such as GAP-43 is a novel mechanism. Whether other examples exist is unknown, but is not an unreasonable speculation given that heterotrimeric G proteins appear to regulate intracellular processes which occur independently of extrinsic signals (Columbo et al., Sdence 255:1695-
  • the inventors added the peptides to neuroblastoma cells briefly permeabilized with phosphatidylcholine as described previously. As expected,
  • Example X demonstrated that GAP-43, an IRP peptide, modulates the activity of G proteins, and through this action, modulates neuronal structural remodeling.
  • the above experiments confirm the su ⁇ rising discovery by the inventors that the amino terminal domain of GAP-43 alone, increases neurite outgrowth, and that this action occurs as a result of the regulation of G-proteins.
  • the invention relates to a particular IRP peptide, comprising an N-terminal amino acid sequence consisting of
  • This N-terminal peptide of GAP-43 has all of the attributes, and is used for all of the purposes previously described for other IRP peptides.
  • This peptide is particularly advantageous in that, because of its small size, it is useful in those situations in which intracellular access requires a smaller peptide.
  • the N-terminal peptide of GAP-43 may be used within a cell to modulate the binding capacity of cellular proteins, including G-proteins, and thereby regulate cellular activity and function in nueuronal and non-neuronal cells.
  • modulatory effects may be either inhibitory or stimulatory, thus acting as agonists, partial agonists, reverse agonists, or antagonists, with respect to a particular physiological effect.
  • the N-terminal peptide of GAP-43 may be used to modulate the stracmral remodeling or plasticity of central or peripheral neuronal cell. It will be appreciated that this modulation may result in either stimulation or inhibition or neuronal growth, depending upon the particular G protein cascade which is modulated.
  • modified N-terminal GAP-43 peptides which also modulate G-protein activity, and thereby neuronal growth. Therefore, another embodiment of the invention relates to the N-terminal peptide of GAP-43 in which d e cysteines at positions 3 and 4 are chemically modified or replaced with other amino acids.
  • these modified peptides include the N-terminal peptide in which cys 3 and cys 4 are palmitoylated or treated witi other sulfhydryl modifiers.
  • Such sulfhydryl modifiers include iodoacetamide, N- ethylmaleimide, and p-chloromercuriphenylsulonate.
  • Other modified peptides include N-terminal peptides of GAP-43 in which cys 3 and cys 4 are replaced with amino acids, such as THR, ASP or GLU.
  • these modified GAP-43 N-terminal peptides may be used in the same manner as other IRP peptides, and in all the methods previously described.
  • the modified compounds may be used to modulate the binding capacity of proteins, such as G proteins, within a cell so as to cause stimulation or inhibition of a desired effect.
  • These compounds may also be used intracellularly to modulate neuronal stracmral remodeling. Modulation of neuronal structual remodeling may result in either stimulation or inhibition of neuronal growth, depending upon the G protein system which is modulated. It is also appreciated that, by using the guidance provided in the foregoing examples, one of skill could develop additional N-terminal peptides of GAP-43 containing other amino acid substitutions, which modulate G protein activity.
  • the invention is directed to pharmaceutical compositions comprising the N-terminal peptide of GAP-43 and the modified derivatives thereof, together with a pharmaceutically acceptable carrier, and optionally comprising one or more therapeutically effective agents.
  • a pharmaceutically acceptable carrier and optionally comprising one or more therapeutically effective agents.
  • the methods of the invention may be carried n vitro, in situ or in vivo, with the latter being most preferred, keeping in mind the general accepted principles of administration which are well known in the art.
  • modulation of neuronal growth provides significant opportunities for the efficacious treatment of neural disorders in mammals, including humans.
  • the above embodiments are especially valuable in preventing, ameliorating, or reversing the effects of neural disease or dysfunction.
  • stimulation of neuronal growth may be especially beneficial in the treatment or prevention of neural developmental abnormalities, neuronal repair from acute injury, or degenerative diseases of the brain.
  • GAP-43 Both Directly Modulates G Protein-Related Second Messenger Systems Within Cells And Amplifies the Sensitivity of G-Protein-43
  • Certain cellular functions are regulated by signal molecules, such as neurotransmitters, hormones and growth factors, which are detected by specific receptors on the plasma membrane of the responding cell. Stimulation of a receptor initiates a cascade of biochemical processes that produce an intracellular signal, mediated by G proteins, which causes a change in the behavior of a cell, for example, secretion of an enzyme, contraction, growth or cell division.
  • signal molecules such as neurotransmitters, hormones and growth factors
  • GAP-43 and G protein-coupled receptors activate G proteins by enhancing guanine nucleotide exchange, and there are sequence similarities in the G protein-interacting regions of the two proteins. This suggests that GAP-43 and receptors might act either antagonistically or synergistically to modulate G protein activity. Studies with purified receptor Go and GAP-43
  • GAP-43 enhances receptor activation of G proteins
  • the inventors conducted several smdies to further investigate the interaction of GAP-43 with receptor-G-protein effector systems within cells.
  • the following experiments show that GAP-43 and receptors act in concert to activate G-protein dependent systems.
  • the experiments involved the incubation of phospholipid vesicles containing purified muscarinic m2 receptor and G 0 , with various concentrations of GAP-43 and the receptor agonist, carbachol.
  • GAP-43 The purification of GAP-43 from neonatal rat brain has been previously described (Strittmatter et al., J. Biol. Chem. 266:22465-22471 (1991)), and the concentration was determined by a dye-binding method (Biorad). Recombinant human muscarinic m2 receptors were isolated from baculoviras- infected insect cells and reconstituted with purified bovine brain G Q into phospholipid vesicles (Parker et al, J. Biol. Chem. 266:519-527 (1991)).
  • GTPase activity at 30°C was measured in the presence of 0.22 nM receptor, 5 nM G 0 , the indicated concentrations of GAP-43, 30 nM ( ⁇ 32 P)GTP, 1.5 ⁇ M GDP, 2 mM MgCl 2 , and 1 mM EDTA with or without 100 ⁇ M carbachol
  • G 0 The activation of G 0 was monitored by the level of steady state GTPase activity (Figure 37).
  • GAP-43 and receptor appear to act in concert, and do not compete with one another. This data provides a molecular basis for the GAP- 43 augmentation of receptor action within cells.
  • GAP-43 augments receptor-stimulated chloride channels in Xenopus oocytes
  • X. laevis oocyte This cell has a well-characterized pathway from transmembrane receptor to G protein to phospholipase C to inositol 1,4,5-triphosphate (IP 3 ) to intracellular Ca ++ release to chloride channel (Moriarty et al, Gprotd s, Iyengar, R. and Birnbaumer, L. eds., New York: Academic Press, Inc., pp. 479-501 (1990)).
  • Stage V and VI oocytes were removed from anesthetized (0.15 % tricaine immersion) female X. laevis (Xenopus I, Ann Arbor, Michigan, USA). The oocytes were defolliculated by incubation with 2 mg/ml collagenase (GIBCO). The cells were stored at 18°C in 96 mM NaCl, 2 mM KC1, 1 mM MgCl 2 , 5 mM Na Hepes, pH 7.6 (ND-96 solution) with 1.8 mM
  • 5HT ⁇ c receptor mRNA was synthesized with T7 RNA polymerase from plasmid MlC2.3-p7 (plasmid generously provided by L. Yu, Indiana University) and 40 nl of a 50 ng/ ⁇ l solution was injected into oocytes 2-4 days prior to further experiments.
  • the 150 ⁇ l bath was perfused with ND-96 containing 0.3 mM CalCl 2 , and acetylcholine (1 ⁇ M) or serotonin (1 ⁇ M) were included in the perfusion for brief periods.
  • GAP-43 was introduced by pressure injection through pipettes of 3 ⁇ diameter, and the volume of injection was pre- calibrated from the size of falling drops of buffer expelled from pipettes of equal diameter with videomicroscopic observation.
  • GAP-43 For the lower concentration of GAP-43, 10 nl of 20 ⁇ M GAP-43 in 100 mM KC1, 5 mM Na Hepes, 0.1 mM DTT, pH 7.5 was injected, resulting in a final estimated concentration of 0.2 ⁇ M from a 200 pmol injection into the 1000 nl oocyte.
  • the higher concentration of GAP-43 was 200 ⁇ M in the pipette, yielding 2 ⁇ M in the cell from a 2 pmol injection.
  • oocytes were injected with two different quantities of GAP-43.
  • the consequent intracellular concentration from the lesser amount is about one-twelfth that of whole neonatal rat brain, and from the greater amount is equal to or slightly less than that in newbom rat brain.
  • An exact comparison of active GAP-43 concentrations is difficult because palmitoylation levels may vary between the oocyte and the brain, and this reversible post- translational modification regulates GAP-43 stimulation of purified G Q (Sudo et al., EMBO J. 77:2095-2102 (1992)).
  • 5HT-induced current in these cells is not altered by buffer injection, but is increased two- to five-fold by the lower GAP-43 concentration and greater than ten-fold by the higher concentration of GAP-43 ( Figure 38).
  • GAP-43 has a direct, transient stimulatory effect on Ca ++ -mediated chloride channel opening under certain conditions (Figure 39).
  • the lower concentration of GAP-43 causes an oscillating inward current of 10-250 nAmps which lasts for 3-10 minutes, when injected 4-10 minutes after a 5HT response, but the same GAP-43 concentration elicits no response from naive oocytes.
  • 5HT and GAP-43 are cross-sensitizers in this system.
  • the reversal potential for the GAP-43 induced current is -21 to -24 mV (not shown), which is the reversal potential for chloride in the oocyte (Kusano et al, J. Physiol 328: 143-170 (1982); Barish, J. Physiol. 342:309-325 (1983)).
  • the response to GAP-43 is abolished if the oocytes are co-injected with EGTA, as predicted for a calcium-mediated event ( Figure 39).
  • Prior IP 3 injection also blocks the response to subsequent high concentrations of GAP-43 for up to two hours (4 of 4 cells, not shown). If the oocyte is allowed to recover from IP 3 for more than six hours, the inward current response to GAP-43 is similar to that of control oocytes. GAP-43 primarily stimulates this system rather than preventing the
  • GAP-43 augments G, inhibition of adenylate cvclase in A431 cells.
  • Non-neuronal cells increase their propensity to form filopodia and decrease their rate of spreading when transfected with GAP-43, and it has been shown that this is dependent on the G protein-interacting domain of GAP- 43. This predicts that G protein-dependent second messenger systems will be altered in GAP-43-expressing cells with a modified mo ⁇ hology.
  • GAP-43 transfectants have different cAMP levels.
  • cAMP levels are low and unaffected by GAP-43.
  • control cell lines accumulate 100 times more cAMP than in the basal state.
  • GAP-43-expressing A431 cells increase cAMP levels only 50-fold in - 25 the presence of forskolin (Fig. 41). This appears to be due to increased G_ function in the GAP-43 cells, because receptor-mediated stimulation of G ⁇ with lysophosphatidic acid (LPA) deceases the level of cAMP in control cells to the level seen in forskolin-treated, GAP-43-expressing cells.
  • LPA lysophosphatidic acid
  • GAP-43 cells contain activated G ; , addition of LPA has no significant inhibitory effect on cAMP levels in the presence of forskolin.
  • the effect of GAP-43 expression on the action of forskolin and LPA is identical to that reported for transfection with mutated Gj a subunits that are constitutively activated ((Wong et al, Nature 351:63 (1991).
  • Prior studies on purified G protein interaction with GAP-43 showed that Gj, but not G reflex is activated by GAP-43.
  • the GAP-43-expressing cells increase cAMP levels only 4.5 fold as compared to 8-fold in the control cells (Fig. 42).
  • LPA decreases isoproterenol- stimulated cAMP levels nearly to basal values in both control and GAP-43- expressing cells.
  • the change in isoproterenol stimulation caused by GAP-43 is similar to the decrease in PGEj stimulation of adenylate cyclase in 3T3 cells transfected with activated a- subunits (Wong, et al Nature 357:63 (1991)).
  • GAP-43 can both augment receptor activation of a G protein transduction cascade, and directly stimulate the same system. It is likely that the molecular mechanism involves GAP-43 stimulation of the a subunit of a G protein, an interaction previously demonstrated for the purified protein. GAP-43 facilitation of receptor agonist action occurs at low GAP-43 concentrations, and therefore, maybe the more prominent effect in vivo. GAP-43 can be considered an intracellular modulator of the sensitivity, or gain, of G protein-coupled receptor transduction. In general, regulation of G protein cascades occurs through transmembrane receptors for extracellular ligands, (Gilman, A. Rev. Biochem.
  • GAP-43 alters growth cone motility by regulating G protein activity.
  • the data also supports the previous demonstration, that GAP-43 can modulate cell shape and, in some settings, increase neurite extension, that G c and GAP-43 are highly concentrated in the growth cone membrane, and that G protein activation state alters neurite extension.
  • GAP-43 could have two actions, one as a direct intracellular regulator of second messenger systems, and a second as a means to increase the sensitivity to receptor stimulation.
  • a means for increasing the sensitivity to receptor stimulation is particularly important in the growth cone, where very shallow gradients of extracellular molecules must be sensed by single filopodia and amplified enormously to alter the behavior of an entire growth cone and axon.
  • the localization of GAP-43 to the neuronal growth cone may reflect a requirement for high level amplification of G protein-mediated signals derived from a single filopodial contact.
  • GAP-43 can be said to increase the "gain" of a G protein- based signal transduction system.
  • GAP-43 provides a means for modulating intracellular receptor-dependent systems which are present in non-neuronal cells.
  • Intracellular receptor-dependent systems have been shown to have a central role in an increasing array of cellular activities, ranging from mating in yeast, to chemically-induced movement in slime molds, to vision, smell, hormone secretion, muscle contraction and cognition in humans.
  • G protein-based signal transduction systems have important roles in the liver (glycogen breakdown), fat cells (fat breakdown), ovarian follicles (synthesis of estrogen and progesterone), kidney cells (water conservation), heart muscle (control of heart rate and force of pumping), smooth muscle cells in blood vessels (contraction, blood pressure elevation), lung (bronchodilation), gastrointestinal tract (gastric motility regulation), and retinal cells (detection of visual signals).
  • GAP-43 may be used to regulate the effects of a variety of receptor-dependent systems, such as those listed above, by modulating receptor stimulation of G proteins.
  • one embodiment of the invention relates to a method for augmenting activation of a desired protein by a receptor, comprising introducing into an environment comprising the protein and receptor, an effective amount of an IRP, such as GAP-43.
  • administration of GAP-43 enables modulation of the duration and thereby, magnitude of receptor activation, and thereby, the resultant intracellular signal.
  • augmentation results in amplification of receptor activation and the intracellular signal.
  • Proteins which are suitable for activation include G proteins, and preferably, G c .
  • receptors include those which activate G proteins in receptor-based systems, and in particular, adrenergic ( ⁇ lt ⁇ and ⁇ , cholinergic ( ⁇ ⁇ , ⁇ 2 , platelet ⁇ 2 , j3,, and 3 2 ), rhodopsin, acetylcholine, including muscarinic and nicotinic, and serotonin.
  • Suitable environments for augmentation of receptor activation include, but are not limited to, cellular compoments of the various organs recited above.
  • a particular aspect of this embodiment provides a means for augmenting receptor-activation of a G protein, and preferably G 0 , by introducing GAP-43 into an environment which consists of a peripheral or central neural cell.
  • GAP-43 modulates structural remodeling of the neuronal cell. It is appreciated by one of skill that the net effect of stracmral remodeling may be either stimulation or inhibition of neuronal growth.
  • Impairment of the function of signal transduction systems contribute to a variety of disorders and diseases involving cellular function, such as cholera, whooping cough, cancer, heart failure, diabetes, and neurological disturbances. Therefore, in another embodiment, administration of GAP-43 would also provide a means for the treatment or prevention of such disorders, in which receptor stimulation of G proteins is impaired. Thus, the amplification of receptor activation effected by G AP-43 would compensate for the deficiency in receptor stimulation, and thus allow the proper functioning of the signal transduction system.
  • Hybridoma ANTI-GAP-43 (H5)
  • Hybridoma, ANTI-GAP-43 (H5)
  • a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28 (4) EPO .

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Abstract

On a cloné l'ADN complémentaire de GAP-43 des mammifères. On décrit les séquences de nucléotides et les séquences d'acides aminés correspondantes pour GAP-43 de l'homme comme du rat. Les séquences inventées, qui sont en pratique pures, peuvent s'exprimer chez des hôtes procaryotes et eucaryotes et servent à surveiller et réguler la croissance neuronale chez des animaux et chez l'homme à des fins tant thérapeutiques que diagnostiques. On décrit de nouveaux peptides ciblant la membrane et pouvant amener une protéine ou un peptide désiré à la membrane cellulaire de cellules, neuronales ou non. On révèle de plus que GAP-43 et ses dérivés peptidiques agissent en tant que protéines et peptides de régulation interne nouveaux des protéines G, telle que Go. En particulier, le domaine terminal amino de GAP-43 module la croissance neuronale grâce à la régulation de Go intervenant dans le cône de croissance. Ces peptides de régulation interne régulent aussi des systèmes intra-cellulaires dépendants de récepteurs en modulant l'activation d'une protéine, telle que la protéine G, grâce à un récepteur, tant dans les cellules neuronales que non neuronales.
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EP1345956A2 (fr) * 2000-10-13 2003-09-24 University of Lausanne Liberation intracellulaire d'effecteurs biologiques par de nouvelles sequences de peptides transporteurs
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WO2000031249A1 (fr) * 1998-11-24 2000-06-02 Pioneer Hi-Bred International, Inc. Promoteurs destines preferentiellement aux racines et utilisations de ces derniers
US6518483B1 (en) 1998-11-24 2003-02-11 Pioneer Hi-Bred International, Inc. Root-preferred promoters and their use
US8183339B1 (en) 1999-10-12 2012-05-22 Xigen S.A. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8236924B2 (en) 1999-10-12 2012-08-07 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8278413B2 (en) 1999-10-12 2012-10-02 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8569447B2 (en) 1999-10-12 2013-10-29 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
EP1345956A2 (fr) * 2000-10-13 2003-09-24 University of Lausanne Liberation intracellulaire d'effecteurs biologiques par de nouvelles sequences de peptides transporteurs
US8080517B2 (en) 2005-09-12 2011-12-20 Xigen Sa Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US8748395B2 (en) 2005-09-12 2014-06-10 Xigen Inflammation Ltd. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US9290538B2 (en) 2005-09-12 2016-03-22 Xigen Inflammation Ltd. Cell-permeable peptide inhibitors of the JNK signal transduction pathway
US9006185B2 (en) 2008-05-30 2015-04-14 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US9180159B2 (en) 2008-05-30 2015-11-10 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory digestive diseases
US9610330B2 (en) 2008-05-30 2017-04-04 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US10023615B2 (en) 2008-12-22 2018-07-17 Xigen Inflammation Ltd. Efficient transport into white blood cells
US8981052B2 (en) 2010-06-21 2015-03-17 Xigen Inflammation Ltd. JNK inhibitor molecules
US9624267B2 (en) 2010-06-21 2017-04-18 Xigen Inflammation Ltd. JNK inhibitor molecules
US9150618B2 (en) 2010-10-14 2015-10-06 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory eye diseases
US10596223B2 (en) 2011-12-21 2020-03-24 Xigen Inflammation Ltd. JNK inhibitor molecules for treatment of various diseases
US10624948B2 (en) 2013-06-26 2020-04-21 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US11779628B2 (en) 2013-06-26 2023-10-10 Xigen Inflammation Ltd. Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of various diseases
US11331364B2 (en) 2014-06-26 2022-05-17 Xigen Inflammation Ltd. Use for JNK inhibitor molecules for treatment of various diseases

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