EP0407543A1 - Mammalian gap-43 compositions and methods of use - Google Patents

Abstract

The mammalian GAP-43 (axonal growth protein) cDNA was produced by cloning. The invention relates to the nucleotide sequences and the corresponding amino acid sequences of human GAP-43 and murine GAP-43. These essentially pure sequences can be expressed in prokaryotes and in host eukaryotes and serve to monitor and regulate neuronal growth in animals, including humans. A new membrane transport peptide is capable of transporting any protein or peptide desired to the cell membrane of neuronal or non-neuronal cells. These essentially pure sequences are useful in therapeutic and diagnostic methods.

Description

TITLE OF THE INVENTION

MAMMALIAN GAP-43 COMPOSITIONS AND METHODS OF USE

Cross-Reference to Related Applications This is a continuation-in-part application of co-pending application Serial Number 401,408, filed September 1, 1989, which is a continuation-in-part of application Serial Number 305,239, filed February 2, 1989, abandoned, which is a continuation-in-part of application Serial Number 288,604, filed December 22, 1988, abandoned, which is a continuation- in-part of application Serial Number 189,223, filed May 2, 1988, abandoned.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the fields of molecular genetics and neurology. More particularly, the invention relates to the cDNA sequence and corresponding amino acid sequence of mammalian GAP-43, a neuronal growth-related protein. 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 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. Description of the Background Art

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 cellul ar proteins , and only a few of these vary such that their levels may be envisioned as directly mediating axonal growth (Skene and Williard, J. Cell. Biol. 89:86 (1981); Benowitz and Lewis, J. Neuroscience 3:2153 (1983); Skene, Cell 37:697 (1984); Meiri, PNAS USA 83:3537 (1986)). Although direct evidence that any of these molecules mediate structural changes is lacking, 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. (Aloyo, J. Neurochem. 41:649 (1983); Akers and Routtenberg, Brain Res. 334:147 (1985)). Furthermore, its level of gene expression correlates well with axonal growth, both in cell culture and in vivo (Basi, Cell 49:785 (1987); Karns, Science 236:597 (1987); Neve, Molec. Brain Res. 2:177 (1987); de la Monte et al.. unpublished).

Absence of repair in the mature human central nervous system (CNS) is a formidable clinical problem. After acute ischemic or traumatic injury histopathological evidence of regeneration is minimal and neurological recovery usually absent or incomplete. On the other hand, neurons of the peripheral nervous system regenerate more predictably, as do CNS neurons of other species, such as goldfish or toad (Skene and Williard, J. Cell. Biol. 89:86 (1981); Benowitz and Lewis, J. Neuroscience 3:2153 (1983)). One explanation for the refractoriness of the mature mammalian CNS neuron might be an irreversible repression of molecules important to growth. In particular, GAP-43 has been suggested as critical to regeneration (Skene, Cell 32:697 (1984)). Evidence for this includes its enrichment in growth cones (Skene, Science 233:783 (1986); Meiri, PNAS USA 83:3537 (1986)) and its minimal expression in the adult as opposed to the perinatal CNS (Skene and Williard, J. Cell. Biol. 89:96 (1981); Karns, Science 236:597 (1987)). Moreover, 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 and Williard, J. Cell. Biol. 89:96 (1981)).

The present inventors have examined the role of GAP-43 in human CNS function and disease. Human GAP-43 cDNA has been cloned, and its developmental and adult distribution examined by assay of post-mortem tissue. In addition to high perinatal expression, 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 expression of GAP-43 even in areas where it is normally low. SUMMARY OF THE INVENTION

Recognizing the potential importance of GAP-43 in mammalian CNS function and disease, the present inventors have succeeded in sequencing the mammalian GAP-43 gene by complimentary DNA (cDNA) cloning. The complete nucleotide sequence of the gene encoding rat GAP-43 and the amino acid sequence have been determined. 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.

Thus, in one embodiment of the invention is provided substantially pure mammalian GAP-43 protein, or a functional derivative thereof. Also provided are 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.

Another embodiment of the invention provides for 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.

Thus, 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.

Employing 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.

Further, 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.

Accordingly, in one exemplary embodiment of the invention is provided 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. It will be appreciated that this embodiment of the invention, and others, may be carried out in vivo, in vitro or in situ, as may be desired.

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. There is thus provided, as a non-limiting example, 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.

Moreover, the inventors have discovered mechanisms by which mammalian GAP-43 expression may be enhanced or, if desired, inhibited. Particularly, it has been discovered that GAP-43 expression is enhanced by nerve growth factor, and that this is inhibited by certain steroids. Inasmuch as 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. Further, 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.

Accordingly, in another aspect, 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.

Thus, in one embodiment, 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. When cells are thus exposed in vitro, it will be possible in another embodiment to introduce such cells into or in close apposition to the location of damaged, diseased or dysfunctioning central or peripheral neural cells with therapeutic effect.

In another embodiment, 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. Alternatively, 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. Similarly, 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.

It has also been discovered that 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.

Thus, 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 or 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. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 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). In brief, 0.5 μg of the GAP43-2 insert, or equivalent amounts of nonspecific DNA, the bacterial plasmid pSP65, were spotted onto nitrocellulose and hybridized with 17.5 μg 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 42ºC for 16 hours. After being washed in standard saline citrate (SSC) (XI), 0.5% SDS at 65°C, the filter was boiled, and the RNA was precipitated with ethanol and translated with rabbit reticulocyte lysate, and the proteins were labeled with [³⁵S]methionine (Pelham et al., Eur. J. Biochem. 67:247 (1976)). Translation products, or products immunoprecipitated with the antibody to GAP-43, were separated on a 12% SDS-polyacryl amide gel. (A) In vitro translation products with (i) no exogenous RNA, (ii) pSP65-selected RNA, (iii and iv) GAP43-2-selected RNA, and (v) poly(A)⁺ newborn brain RNA (newborn). (B) Immunoprecipitations by antibody to GAP-43 of the translation products of (A), as described for (A) except for the fourth lane, which shows immunoprecipitation of the translation product after having preabsorbed the GAP-43 antibody with GAP-43 protein, prepared as in Snipes et al .. Soc. Neurosci. Abstr. 12:500 (1986).

Figure 2. 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 and Hoffman, Gene 25:263 (1983), ligated to EcoRI linkers, and ligated into the EcoRI site of the lambda phage cloning vectors, λgt10 and λgt11. The longest clone identified, GAP43-2, and two phage with smaller inserts, were identified from about 5 × 10⁴ plaques in the λgtll library after induction with isopropyl β-D-thiogalactopyranoside (IPTG), by using the rabbit antibody to GAP-43, followed by alkaline phosphatase-conjugated antibody to rabbit immunoglobulin G (Promega Biotec). The cDNA inserts were subcloned into the EcoRI sites of M13 mpl8. Initial DNA sequence analysis of the two shorter clones revealed that they were included within the longest. 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 λgtll 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. E, EcoRI; M, Mspl; V, PvuII; H, Haelll; P, Pstl; S, Sau3A. The arrow between nucleotides 100 and 101 indicates the boundary between the first and second exons; the arrow between nucleotides 664 and 665 indicates the start of the third exon. Figure 3. Regulation of GAP-43 expression in PC12 cells. PC12 cells were passaged in RPMI medium containing 10% horse serum and 5% fetal bovine serum. Forty hours after plating the cells, the medium was changed to include the different additives. After 4 days, the cells were photographed (A), then RNA was isolated from each cell culture. RNA (10 μg per sample) was denatured and run on a 1.2% agarose-formaldehyde gel, transferred to a GeneScreen nylon filter, bound to the filter by ultraviolet cross-linking, and probed with ³²p- labeled GAP43-2 (B). The final wash was SSC (×0.2), 0.1% SDS at 65°C. The additives included were (i) none, (ii) 50 ng of NGF per milliliter, (iii) 10^-3M dibutyryl cAMP, and (iv) 50 ng of NGF per milliliter and 10^-3M dibutyryl cAMP. The last lane is 10 μg of RNA from newborn brain run as a positive control for the blotting and hybridization procedure.

Figure 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 18:5294 (1979). Each RNA (10 μg) was denatured, underwent electrophoresis in a 1.2% agarose-formaldehyde gel, and was transferred to nitrocellulose. The filter was hybridized overnight at 42ºC with the EcoRI insert from λgtll GAP43-2 labeled with deoxycytidine 5'-[α-³²P]triphosphate by nick translation. The final wash was done in SSC (x0.2), 0.1% SDS at 65°C. 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. The positions of the 18S and 28S ribosomal RNA are shown at the right. Below is hybridization of the same filter with a cDNA probe encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Piechacyk et al., Cell 42:589 (1985)).

Figure 5. (A) 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 predicted by fold program of Zuker and Steigler, Nucl. Acids Res. 9:133 (1981).

Figure 6. Northern blot showing the regional restriction of GAP-43 expression with maturation. Ten μg 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.

Figure 7. Northern blot showing that GAP-43 expression increases in the wake of an ischemic event. (A) Ten μg 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 (All). (B) Ten μg of RNA from Area 3,1,2,5 from three patients, all run 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. Figure 8. In situ hybridization reveals increased GAP-43 expression in regions adjacent to infarcts. (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 (x160). (A2) Normal adjacent cortex with abundant histopathologically intact neurons (×300). (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) (×12). (C) In normal visual cortex GAP-43 expression is restricted to a few scattered neurons by darkfield examination (arrowheads). (D) In the infarcted cortex there is no specific binding of the anti sense GAP-43 probe. The large bright foci are from areas of coagulative necrosis which also label nonspecifically with the sense probe (G). In contrast, in the adjacent intact cortex numerous neurons express GAP-43 (E-brightfield and F-darkfield labeled with antisense probe). (H) A control brightfield and (I) darkfield labeled with the sense probe showing absence of specific GAP-43 binding (C-I ×160).

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 Purkinje cell and outer granule cell layers. (C) A section adjacent to B hybridized with the sense strand probe as control (all ×315).

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: 1A, 406; IB, 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).

Figure 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 immunofluorescence, or both. The intact GAP-43 gene (GAP) was significantly membraneassociated, as were GAP constructions lacking substantial portions of exon 2 (GAP(-intern.)) or the carboxy-terminus region of the GAP-43 gene (GAPtag). However, when the nucleotides encoding the first four amino acids of GAP-43 were deleted (GAP(- 1-4)), the expressed protein fragment was not membrane-associated.

Point mutations were introduced into the sequences encoding the cysteines at positions three (C3) or four (C4) of the first exon, to result in expression of alanine in the resulting protein. Mutation of either C3 (GAP *C₃) or C4 (GAP *C₄) resulted in reduced membrane levels (+/-) as compared to intact GAP-43. Reduction was especially marked when C4 was altered. Mutation of both C3 and C4 (GAP *C_3,4) eliminated membrane association altogether.

Transient expression of the gene encoding chloramphenicol acetyl transferase (CAT) produced no membrane-associated protein, as expected for this normally cytosolic enzyme. When the nucleotide sequence encoding the ten amino acids of the first GAP-43 exon was ligated to the amino-terminus end of the CAT gene (GAP(1-10)CAT), the expressed protein was membraneassociated.

Figure 12. Western blot showing that normal GAP-43 has both a membrane and a cytosolic component (M = membrane; C = cytosolic) when transfected into CHO cells. Mutation of the nucleotide sequence encoding the third or fourth cysteines (C-3 or C-4) of the first GAP-43 exon interfered with the membrane-binding component, while mutation of both cysteines (C-3,4) abolished membrane binding completely. Control cells (CON) had no GAP-43; Brain membranes (BR) had GAP-43 of the same molecular weight as that from the transfected gene.

Figure 13. Map of the rat GAP-43 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).

B. Intron-exon boundaries and 3' polyadenylation site. The exons and adjoining regions were sequenced and intron-exon boundaries were determined by comparison to the cDNA sequence as described herein, taking the best fit to consensus splice sites (Mount, Nucl. Acids Res. 10:459-472 (1982)). The major polyadenylation site was determined by RNAse protection. The putative polyadenylation signal and a tandem pair of a consensus motif often found immediately 3' of utilized polyadenylation signals (McLauchlan et al., Nucl. Acids Res. 13:1347-1368 (1985)) are underlined.

Figure 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.

Figure 15. Mobility shift in restriction fragments induced by H-DNA. A schematic map of the 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). Below are representations of the GAP-43 promoter fragments liberated by digestion of plasmid bsl.5RIX4 with the following enzymes: 1) Sspl, 603 bp; 2) Xbal/Sspl, 560bp; 3) Smal/Sspl, 490bp; 4) Sspl/Nhel, 409bp; 5) Sspl/Nsil, 284bp (contains region I), 319bp (contains regions II and III; 6) Sspl/Accl, 314bp (region I), 289bp (regions II and III); 7) Xbal/Nhel, 360bp; and 8) Smal/Nhel, 295bp.

Figure 16. Partial protein sequence for p34 and p38.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following description, reference will be made to various methodologies known to those of skill in the art of molecular genetics and neurology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full.

Standard reference works setting forth the general principles of recombinant DNA technology include Watson, J.D. et al . , Molecular Biology of the Gene. Volumes I and II, The Benjamin/Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987); Darnell, J.E. et al ., Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986); Lewin, B.M., Genes II. John Wiley & Sons, publishers, New York, N.Y. (1985); Old, R.W., et al.. Principles of Gene Manipulation: An Introduction to Genetic Engineering. 2d edition, University of California Press, publisher, Berkeley, CA (1981); and Maniatis, T., et al.. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, NY (1982).

By "cloning" is meant the use of in vitro recombination techniques to insert a particular gene or other DNA sequence into a vector molecule. In order to successfully clone a desired gene, 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.

By "cDNA" is meant complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase). Thus a "cDNA clone" means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.

By "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 Cloning: A Laboratory Manual, supra. Generally, 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.

By "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. Thus, by "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. Such DNA expression vectors include bacterial plasmids and phages. Preferred for the purposes of the present invention is the lambda gtll expression vector.

By "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. By "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. As used herein, 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. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Penn. (1980).

Similarly, 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, T., et al .. supra, 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. The precise nature of the 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. 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.

If desired, 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. Thus, by retaining the 3'-region naturally contiguous to the DNA sequence coding for the protein, 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 (such as a promoter region sequence and a GAP-43 encoding sequence) 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. Thus, a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence.

Thus, to express the protein, transcriptional and trans!ational signals recognized by an appropriate host are necessary. 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. Bacillus. Streptomvces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Other enterobacterium such as Salmonella tvphimurium 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 rep! icon and control sequences in the expression plasmid.

To express the GAP-43 protein (or a functional derivative thereof) in a prokaryotic cell (such as, for example, E. coli. B. subtilis, Pseudomonas, Streptomvces, etc.), it is necessary to operably link the GAP-43 encoding sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the ø-lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pBR325, etc. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (PL and PR), the trp, recA. lacZ, lad, and gal promoters of E. coli, the α-amylase (Ulmanen, I., et al.. J. Bacteriol. 162:176-182 (1985)) and the σ-28-specific promoters of B. subtilis (Gilman, M.Z., et al.. Gene 32:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, T.J., In: The Molecular Biology of the Bacilli. Academic Press, Inc., NY (1982)), and Streptomvces promoters (Ward, J.M., et al .. Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters are reviewed by Glick, B.R., (J. Ind. Microbiol. 1:277-282 (1987)); Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann. Rev. Genet. 18:415-442 (1984)). Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream of the geneencoding sequence. Such ribosome binding sites are disclosed, for example, by Gold, L., 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-trans!ational 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 SP2/0-AG14 or the myeloma P3×63Sg8, 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-trans!ational 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.

For a mammalian host, many possible vector systems are available for the expression of GAP-43. A wide variety of 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. Alternatively, 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. Of interest are 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. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of the desired proteins in yeast. 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. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

Production of 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. Thus, in one embodiment, sequences encoding GAP-43 may be operably linked to the regulatory regions of the viral polyhedrin protein (Jasny, Science 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. When live insects are to be used, caterpillars are presently preferred hosts for large scale GAP-43 production according to the invention.

As discussed above, expression of the GAP-43 protein in eukaryotic hosts requires the use of 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, D., et al.. J. Mol . APPI. Gen. 1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell 31:355-365 (1982)); the SV40 early promoter (Benoist, C, et al., Nature (London) 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston, S.A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic promoter and a DNA sequence which encodes the GAP-43 protein (or a functional derivative thereof) does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in the same reading frame as GAP-43 encoding DNA sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the GAP-43 encoding sequence).

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 integration of the introduced sequence into the host chromosome.

In one embodiment, 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 prototrophy to an auxotropic 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 introduced 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, H., Mol . Cel. Biol. 3:280 (1983).

In a preferred embodiment, the introduced sequence will be incorporated into 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, Col El , pSC101, pACYC 184, πVX. Such plasmids are, for example, disclosed by Maniatis, T., et al . (In: Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). Bacillus plasmids include pC194, pC221, pT127, etc. Such plasmids are disclosed by Gryczan, T. (In: The Molecular Biology of the Bacilli. Academic Press, NY (1982), pp. 307-329). Suitable Streptomvces plasmids include pIJ101 (Kendall, K.J., et al.. J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as ϕC31 (Chater, K.F., et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John, J.F., et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978)).

Preferred eukaryotic plasmids include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids are well known in the art (Botstein, D., et al., Miami Wntr. Svmo. 19:265-274 (1982); Broach, J.R., In: The Molecular Biology of the Yeast Saccharomvces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, J.R., Cell 28:203-204 (1982); Bollon, D.P., et al., J. Clin. Hematol . Oncol. 10:39-48 (1980); Maniatis, T., In: Cell Biology: A Comprehensive Treatise. Vol. 3. Gene Expression, Academic Press, NY, pp. 563-608 (1980)).

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, 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., Science 240(4858): 1538 (1988)), etc.

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-gal actosidase, or any desired homologous or heterologous protein or peptide. Methods for producing such fusion proteins are taught, for example, Bai, D.H. et al., J. Biol. Chem. 261:12395-12399 (1986), or Huynh, T.U. et al., "Construction and Screening cDNA Libraries in λgtlO and λgt11," in DNA Cloning Techniques: A Practical

Approach. D. Glover (ed.), IRL Press, Oxford, 1985, pp. 49-77.

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. For example, 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. Alternatively, 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. Such antibodies may be obtained by well-known methods, some of which as mentioned hereinafter. Thus, for example, 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. The term "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. For example, 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.

In the most preferred method, 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 et al.. Eur. J. Immunol. 6:292 (1976); Hammer! ing et al .. In: Monoclonal Antibodies and T-Cell Hybridomas. Elsevier, N.Y., pp. 563-681 (1981)). In general, 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. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands, J.R., 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.

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.

Standard reference works regarding the general principles of immunology include Klein, J., Immunology: The Science of Self-Nonself Discrimination. John Wiley & Sons, Publisher, New York (1982); Kennett, R., et al. , eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses. Plenum Press, Publisher, New York (1980); Campbell, A., "Monoclonal Antibody Technology," in, Burdon, R., et al. , eds., Laboratory Techniques in Biochemistry and Molecular Biology. Volume 13, Elsevier, Publisher, Amsterdam (1984).

By "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.

The isolation of other hybridomas secreting monoclonal antibodies of the same specificity as those described herein can be accomplished by the technique of anti-idiotypic screening. Potocmjak, et al., Science 215:1637 (1982). Briefly, 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). By using the antiidiotypic 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. Thus, by using anti-bodies to the epitopic determinants of a monoclonal antibody, it is possible to identify other clones expressing antibodies of identical epitopic specificity. In antibodies, idiotypic determinants are present in the hypervariable region which binds to a given epitope.

Accordingly, 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.

For replication, 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 supernatants 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. In addition, the antibodies in these immunoassays can be detectably labeled in various ways.

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. This enzyme, in turn, when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected as, for example, by spectrophotometric or fluorometric means. Examples of 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-gal actosidase, 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 ³H, ¹²⁵I, ³²p, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe and ⁷⁵Se.

It is also possible to detect the binding of the detectably labeled antibodies of the present invention by labeling the antibodies with a fluorescent compound. When a fluorescently labeled antibody is exposed to light of the proper wave length, its presence then can be detected due to the fluorescence of the dye. Among the most commonly used fluorescent labeling compounds are 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 ¹⁵²Eu, 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).

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. Examples of particularly useful chemiluminescent labeling compounds are luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the antibodies of the present invention. 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 luminescence. Important bioluminescent compounds for purposes of labeling include luciferin, luciferase and aequorin.

The antibodies and substantially purified antigen of the present invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being com-, partmentalized 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.

The types of assays which can be incorporated in kit 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 (EIA), enzyme-linked immunosorbent assays (ELISA), and immunometric, or sandwich, immunoassays.

By the term "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.

Forward sandwich assays are described, for example, in United States Patents 3,867,517; 4,012,294 and 4,376,110. Reverse sandwich assays have been described, for example, in United States Patents 4,098,876 and 4,376,110. In the preferred mode for preforming the assays it is important that certain "blockers" be present in the incubation medium (usually added with the labeled soluble antibody). The "blockers" are added to assure that non-specific proteins, protease, or human antibodies to mouse immunoglobulins present in the experimental sample do not cross-link or destroy the antibodies on the solid phase support, or the radio!abeled indicator antibody, to yield false positive or false negative results. The selection of "blockers" therefore adds substantially to the specificity of the assays described in the present invention.

It has been found that a number of nonrelevant (i.e. nonspecific) antibodies of the same class or subclass (isotype) as those used in the assays (e.g. IgG₁, IgG_2a, IgM, etc.) can be used as "blockers." The 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. In addition, the 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 physiological pH ranges. Somewhat less preferred buffers are inorganic buffers such as phosphate, borate or carbonate. Finally, known protease inhibitors should be added (normally at 0.01-10 microgs/ml) to the buffer which contains the "blockers."

There are many solid phase immunoadsorbents 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 eovalently 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.

For in vivo, in vitro or in situ diagnosis, 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 anti-bodies is diethylenetriaminepentaacetic acid (DTPA). Typical examples of metallic cations which are bound in this manner are: ^99mTc, ¹²³I, ¹¹¹IN, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu. ⁶⁷Ga and ⁶⁸Ga. The antibodies of the invention can also be labeled with nonradioactive isotopes for purposes of diagnosis. Elements which are particularly useful in this manner are ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.

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 with epitopes reactive with the antibodies of the present invention.

When used for immunotherapy, the antibodies of the present invention may be unlabeled or labeled with a therapeutic agent. Examples of 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., Science 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 Corvnebacterium 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.

Examples of radioisotopes which can be bound to the antibodies of the present invention for use in immunoth_erapy are: ¹²⁵Um, ¹³¹I, ⁹⁰γ, ⁶⁷Cu, ²¹⁷Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc and ¹⁰⁹Pd.

Of course, 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.

The choice of 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.

Examples of 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 Biol. 107(6/3): Abstr. No. 3112 (1988), as well as known brain-specific receptors such as those for dopamine. In this regard, it will be appreciated that the ligand may itself be an antibody or fragment specific for the receptor, to which may be conjugated the antibody of the invention.

Moreover, those of skill may find it particularly desirable to employ antibody fragments of the invention (such as, for example, Fab or F(ab')₂ fragments), 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.

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 therapeutically 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.

It is also possible to utilize 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.

Another preferred manner in which the antibodies, and preferably, the GAP-43 encoding nucleotide sequences (and their functional and chemical derivatives) may be introduced into neural cells for diagnostic or therapeutic purposes is by the use of viral, including retroviral, vectors. As an example of suitable viruses may be mentioned the various herpes viruses. Suitable retroviruses include human immunodeficiency virus (HIV). Other suitable viruses and retroviruses are well known to those of skill. The use of viral vectors for introduction of genes into mammalian cells is reviewed, for example, in Varmus, Science 240(4858): 1427

(1988); Eglitis et al., BioTechnioues 6,7: 608 (1988);

Jaenisch, Science 240(4858): 1468 (1988); and Bernstein et al., Genet. Eng. (N.Y.) 7:235 (1985).

For the purposes of the present invention, it may be preferred to employ an attenuated viral or retroviral strain. Thus, for example, it is possible to use as vectors for the antibodies or DNA sequences of the invention retroviruses having attenuated cytopathicity, such as HIV-2_ST (Kong et al., Science 240(4858): 1525 (1988)) or HIV-2_UC1 (Evans et al., Science 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. 2(14): 2970 (1988). Those of skill will be able to target different neural populations having known susceptibilities to viruses by the exercise of routine skill. For example, CD4 is known to have a variant transcript in the human brain, with its highest content in forebrain (Maddon et al., Cell 47: 333 (1986). Ideally, then, the choice of a gene delivery system will be made by those of skill, keeping in mind the objectives of efficient and stable gene transfer, with an appropriate level of gene expression, in a tissue-appropriate manner, and without any adverse effects. See, for example, Wolff et al., Rheum. Dis. Clin. North Am. 14(2): 459 (1988). With respect to delivery to a central nervous system target, many viral vectors, including HIV, offer the advantage of being able to cross the blood-brain barrier (Johnson et al., FASEB J. 2(14): 2970 (1988)).

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 therapeutical ly 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 skill in further elucidating the mechanisms of neuron structure and function.

The preclinical and clinical therapeutic use of the present invention in the treatment of neurological disease or disorders will be best accomplished by those of skill, employing accepted principles of diagnosis and treatment. Such principles are known in the art, and are set forth, for example, in Petersdorf, R.G. et al., eds., Harrison's Principles of Internal Medicine, 10th Edition, McGraw-Hill, publisher, New York, N.Y. (1983), especially at Part 6, Section 11 of that work, entitled "Disorders of the Central Nervous System."

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.

The pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, 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.

In addition to the pharmacologically active compounds, 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. Preferably, 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^), 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. Generally, 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 anti-bodies can be administered parenterally by injection or by gradual profusion over time. The antibodies of the present invention also can be administered intravenously, intraparenterally, intramuscularly or subcutaneously.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, drageemaking, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resul ting mixture and processing the mixture of granul es, 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 sorbitol, 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 carboxymethyl cellulose, and/or polyvinyl pyrrolidone. If desired, 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. For this purpose, concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrol idone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropymethylcellulose 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. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin. In addition, 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. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycol s, or paraffin hydrocarbons.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds in watersoluble form, for example, water-soluble salts. In addition, 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 include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, 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. Thus, 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. In another embodiment, 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 may in turn be used to detect or measure antibody to GAP-43 in a sample, such as cerebrospinal fluid, serum or urine. Thus, 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. It will be appreciated that immunoreactive fractions and immunoreactive analogues of GAP-43 also may be used. By the term "immunoreactive fraction" is intended any portion of the GAP-43 antigen which demonstrates an equivalent immune response to an antibody directed against GAP-43. By the term "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.

In yet another aspect of the present invention, it has been found that the GAP-43 protein contains a novel membranetargeting 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 membranetargeting 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. Experiments illustrating this aspect of the present invention are presented in detail in Example VI of the specification. According to the 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 preceeding 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.

The manner and method of carrying out the present invention may be more fully understood by those of skill by reference to the following examples, which examples are not intended in any manner to limit the scope of the present invention or of the claims directed thereto.

EXAMPLE I Cloning of the cDNA for Rat GAP-43

A cDNA library was generated from RNA of rat dorsal root ganglia from embryonic day 17 and cloned into the λgt11 expression vector (Huynh et al.. in "DNA Cloning A Practical Approach," D.M. Glover, Ed. (IRL Press, Washington, D.C., 1985) pp. 49-78). Three presumptive GAP-43 clones were identified with the antibody to GAP-43 described by Snipes et al.. Soc. Neurosci. Abstr. 12:500 (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. The specificity of the immunoprecipitation was demonstrated by competition with unlabeled, purified GAP-43. For additional confirmation, a peptide prepared by cyanogen bromide cleavage of purified GAP-43 was sequenced. The sequence, Arg-X-LysGln-Val-Glu-Lys-Asn-Asp-Glu-Asp-GIn-Lys-Ile, is completely included within the predicted open reading frame of GAP43-2. (The X represents a cycle of sequencing at which the identity of the amino acid could not be determined with certainty.)

The complete nucleotide sequence of GAP43-2 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 β-galactosidase gene of λgt11. (A cDNA for rat GAP-43 was obtained independently by J.H.P. Skene and his colleagues (G. Basi, R. Jacobson, I. Virag, J.H.P. Skene, personal communication). Copies of the sequences were exchanged. The predicted amino acid sequence of the present invention agrees perfectly with that provided by J.H.P. 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. However, the information is insufficient to make this assignment unequivocally, and, therefore, the second methionine (amino acid 5) might play this. role. The predicted composition of GAP-43 is highly polar, without evident transmembrane domains or potential N-l inked 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-polyacryl amide gels (Skene and Williard, J. Cell. Biol. 89:86 (1981), ibid., p. 96). The molecular size has been uncertain because the apparent molecular size of GAP-43 depends on polyacryl amide concentration (Jacobson et al., J. Neurosci. 6:1843 (1986)), suggesting that this protein falls in the category of proteins that migrate anomalously on SDS-polyacryl amide gels (Banker et al.. J. Biol. Chem. 247:5856 (1972); Persson et al.. Science 225:687 (1984); Smart et al.. Virology 112:703 (1981)). This property is unlikely to be due to posttransl ational modification since the in vitro translation product has a mobility similar to that of native GAP-43 (Fig. 1).

To collect information concerning the SDS-polyacryl amide gel migration properties of the protein encoded within the putative open reading frame, 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 Acids Res. 12:7035 (1984). An 800-base 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 poly! inker 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.

Evidence for the belief that GAP-43 is important to the function of growth cones includes enrichment of the protein in growth-cone membranes (Meiri et al., PNAS USA 83:3537 (1986); Skene et al., Science 233:783 (1986)) and increased transport of the protein in developing and regenerating nerves (Skene and Williard, J. Cell. Biol. 89:86 (1981), ibid., p. 96). To investigate whether 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). These agents act by different mechanisms in inducing neurite outgrowth (Gunning et al., J. Cell. Biol. 89:240 (1981)). Concomitant with the neurite growth induced by either agent is an increase in GAP-43 mRNA levels, with the largest increase in cells exposed to both agents (Fig. 3).

To determine the pattern of expression of the GAP-43 gene during normal development, total cellular RNA was isolated from brain, dorsal root ganglia, heart, and liver of embryonic day 13, embryonic day 17, newborn, and adult rats. 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, largemolecular-size bands may correspond to unspliced precursor molecules, since the present inventors have discovered that the genomic GAP-43 gene contains intronic sequences. The 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.

In neural tissues, 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)). However, the significant amount of 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. Neurosci. 6:1843 (1986)). The persistence of GAP-43 expression suggests an ongoing role in the adult nervous system. The properties of B-50 and F1, phosphoproteins electrophoretically and antigenically indistinguishable from GAP-43, have been assessed in adult neuronal tissue. These proteins serve as substrates for a protein kinase C-like enzyme, and their phosphorylation is regulated by neuropeptides, neurotransmitters, and during the course of long-term potentiation (Jacobson et al., J. Neurosci. 6:1843 (1986); Aloyo et al., J. Neurochem. 41:649 (1983); Zwiers et al .. Progr. Brain Res. 56:405 (1982)). It is not known whether 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 i nclude an ongoing rol e in synaptic turnover (Cotman et al .. Science 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, Science 220:91 (1983)). EXAMPLE II

Cloning of the cDNA for Human GAP-43

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 2×2×0.5 cm, obtained from specific regions were snap-frozen in isopentane (2-methyl butane) cooled with dry ice and then stored at -90ºC. 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 formalinfixed, paraffin-embedded tissue immediately adjacent to the frozen blocks. cDNA cloning

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 ³²P-labeled rat GAP-43 cDNA probes (Karns et al.. Science 236:597 (1987)). Hybridization was for 16 hr at 42ºC in 4× Standard Saline Citrate (SSC), 0.8x Denhardt, 10% dextran sulfate, 40% formamide, 20 μg herring sperm DNA per ml, 7 mM Tris (pH 7.6), 1% SDS, and probe at 10⁶ cpm/ml. Filters were washed extensively in 2× SSC at room temperature, 1× SSC at 53ºC and again 1× SSC at 60ºC before autoradiography. Positive clones were subcloned into both pGem-3Z (Promega Biotec) and M13 mp18 and sequenced by the chain-termination method (Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977)). DNA sequences were analyzed by UWGCG (University of Wisconsin Genetics Computer Group) software package.

Northern blot analysis

Total cellular RNA was isolated from autopsied human tissues by the guanidium thiocyanate procedure (Chirgwin et al.. Biochemistry 18:5294 (1979)). For Northern blots, 10 μg of total RNA from each tissue were denatured and run on a 1.2% agarose-formaldehyde gel, transferred to Genescreen (New England Nuclear) or Nytran (S & S) in 10× SSC, UV crosslinked, and hybridized overnight at 42°C with randomly primed (Feinberg and Vogelstein, Anal. Biochem. 132:6 (1983)) human GAP-43 cDNA clone Cla. Filters were finally washed to 1× SSC with 0.1% SDS at 60ºC. After probing with Cla, the filters were stripped and reprobed with a rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA probe (Piechaczyk et al., Cell 42:589 (1985)) as control.

In situ hybridization

Cryostat sections of human brain tissue were fixed in 4% paraformaldehyde, treated with 0.3% Triton X-100 followed by 1 μg/ml proteinase K, acetylated, and pre-hybridized in 50% formamide/2x SSC. Hybridization using 2 × 10⁶ cpm per slide of ³⁵S-labeled antisense or sense riboprobe (Melton et al., Nucleic Acids Res. 12:7035 (1984)) was performed in a humidified chamber for 5 hours at 50°C. The tissue sections were then washed in 2× SSC with 10 mM dithiothreitol initially containing 50% formamide, then 50% formamide plus 0.1% TritonX 100. Single stranded RNA was removed by treatment with 50 μg/ml RNAase A. The sections were further washed in 2× SSC with ImM DTT for 2 hours, then dehydrated through graded alcohols containing 0.3 M ammonium acetate. The radioactive signal was detected using NTB2 Kodak emulsion. Emulsioncoated slides were counterstained with hematoxylin. RESULTS

Human GAP-43 is homologous to a mouse calmodul in binding protein

As described in Example I, 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. The identity of GAP-43 with that of a neural specific mouse calmodul in-binding protein, termed P-57, was described by Cimler et al.. J. Biol. Chem. 262:12158 (1987), so that sequence is also aligned in Figure 5B. P-57 has been described as a neural -specific calmodul in-binding protein with the unusual property that it releases, rather than binds, calmodulin as calcium levels rise (Andreasen et al., Biochemistry 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. Sci. USA 83:3228 (1986); Shaw and Kamen, Cell 46:659 (1986)).

GAP-43 expression persists in discrete regions of the adult human brain

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-43 was uniformly and robustly expressed throughout the brain as assessed by Northern blots. The regions examined included the cerebellum, temporal cortex, temporal association cortex, frontal cortex, orbital frontal region (Area 11), hippocampus, visual cortex (Area 17/18), and spinal cord, some examples of which are shown in Figure 6. In contrast to the brain, levels in the spinal cord were low at these ages, which may be related to earlier maturation of this region (Anand and Hickey, New Enol. 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. For example, in three brains, levels comparable to the neonate were found in Broadman's Area 11 (orbital frontal gyrus) and much lower levels expressed in the visual cortex (Area 17/18) . Level s were consistently low in the hippocampus. The latter is of interest because the adult rat hippocampus is enriched in GAP-43. A similar distribution has been reported recently by Neve et al.. Molec. Brain Res. 2:177 (1987).

Renewed GAP-43 expression after ischemic injury

Brain tissue was examined from two patients with small clinically silent infarcts, both occurring 10 to 14 days antemortem. The histopathological features that characterized these subacute infarcts included the following: [1] a sharp delineation between the infarcted and the intact tissue; [2] loss of neurons; [3] infiltration of necrotic tissue by mononuclear inflammatory cells and lipid-laden macrophages; and [4] activation of fibrous astrocytes along the edge of the infarct. In one patient 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). These observations suggest that GAP-43 increases within days following ischemic infarction.

As described above, 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. To examine this hypothesis, in situ hybridization was used to study the distribution of GAP-43 expression in the region of infarction. The detailed cellular anatomy of GAP-43 expression is presented in Example III. Throughout most regions of the adult cerebral cortex, including the visual cortex (Area 17), as might be predicted from the Northern analysis, only a few scattered cells expressed GAP-43 (Figure 8C). In the infarcted region of visual cortex, no specific GAP-43 expression was found, although frankly necrotic regions bound both antisense and sense probes non-specifically (Figure 8D, 8G). On the other hand, in the adjacent morphologically normal Area 17 (Figure 8A2) essentially all neurons evidenced GAP-43 expression (Figures 8E, 8F), confirming that regions adjacent to infarcted tissue are the source of the increased GAP-43 expression.

The effect of transient ischemia without infarction upon GAP-43 expression was also investigated. Neurons in certain regions of the brain, such as the cerebellar cortex and Sommer's sector of the hippocampus, manifest selective vulnerability to ischemic and hypoxic insults, particularly when due to hypoperfusion (Brierley and Graham, In: Greenfield's Neuropathology. Fourth Edition, J.H. Adams, Corsellis, J.A.N., and Duchen, L.W., Eds., Edward Arnold, London, 1984, pp. 125-156). This type of injury may be transient and full recovery may ensue within weeks. Using in situ hybridization, the cerebellar cortex of one patient who sustained a cardiac arrest with attendant global anoxia was examined. The patient died several days later, and at autopsy there was evidence of anoxic encephalopathy without infarction. This was manifested by the presence of numerous scattered pyknotic (dark and shrunken) or hydropic (swollen) neurons and vacuolation of the neuropil in the cerebral cortex, hippocampus and cerebellum. In the cerebellum, the ischemic 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). DISCUSSION

Growth cones are nerve terminal structures shared by developing and regenerating nerves (e.g., Ramon y Cajal, "Degeneration and Regeneration of the Nervous System," Oxford University Press, London (1928); Kater and Letourneau, "Biology of the nerve growth cone," Alan R. Liss, Inc., New York (1985)). They include machinery for motility and transduction of local information and have a protein constituency determined by transport from the cell soma. 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)). Thus 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 calmodul in-binding protein which has the unusual property of releasing calmodul in 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)). As suggested by the above authors, one notion for its role in the growth cone might be that it regulates calmodul in activity, and that it does so by releasing it in focal cellular domains. Thus, the affinity of GAP-43 for calmodul in would diminish when calcium rises, for example after an action potential (Belardetti et al.. Proc. Natl. Acad. Sci. 83:7094 (1986)). The calmodul in-dependent activities of the growth cone could thereby be regulated within the immediate vicinity of calcium entry.

GAP-43 in the adult

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. One possibility is that 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. The other function proposed for GAP-43 is learning, because its phosphorylation state changes in the wake of long-term potentiation (Nelson and Routtenberg, Exp. Neurol . 89:213 (1985)). In fact, structural remodeling may be a facet of long-term learning (Chang and Greenough, 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, Science 220:91 (1983)). Thus, one intriguing possibility is that neurons of the adult human brain that use structural pl asticity for l ong-term l earning are those that express GAP-43. This possibility has been discussed by several investigators, including Nelson and Routtenberg, Exp. Neurol . 89:213 (1985), Jacobson et al., J. Neurosci. 6:1843 (1986), and Neve et al., Molec. Brain Res. 2:177 (1987), and the present work is consistent with these suggestions. This proposal will require close correlation of various disease states with GAP-43 expression.

GAP-43 and repair in the adult CNS

The restraints upon recovery from CNS injury are poorly understood. Whereas severance of a peripheral nerve of mammals or even central nerves of amphibia may be followed by full repair, no recovery follows such injury in the central nervous system of mammals. The adult neuron does retain the ability to grow, since central nerves will elongate long distances after axotomy so long as they are provided with a peripheral nerve sheath as a guide (Benfey and Aguayo, Nature 296:150 (1982)). The failure to repair has been attributed to different regulatory controls over a group of axonally transported proteins in the central nervous system as opposed to the peripheral nervous system. Specifically, 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. Whether 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. Alternatively, 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. For example, these might include the release of excitatory amino acids and consequent N-methyl-D-aspartate (NMDA) 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, J. Neurosci. 4:1884 (1984)).

EXAMPLE III

Detection of GAP-43 Expression Employing Antibody

Directed Against Purified GAP-43 Protein

Previous immunohistochemical studies have suggested GAP-43 to be restricted to neurites, especially in the developing nervous system (Benowitz et al., J. Neurosci. 8:339-352 (1988); Gispen et al.. Brain Res. 328:381-385 (1985); Meiri et al.. Proc. Natl. Acad. Sci. USA 83:3537-3541 (1986); Skene et al.. Science 233:783-786 (1986)). In the present example, using in situ hybridization and a novel antibody to GAP-43 that reveals perikaryal staining, the inventors identify the cell populations that express GAP-43, and demonstrate that in the adult brain the widespread GAP-43- immunoreactive neurites emanate from a relatively small population of neurons, most of which are regionally restricted.

METHODS In situ hybridization

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 μg/ml proteinase K, acetylated, and pre-hybridized in 50% formamide/2× standard saline citrate (SSC). The probe was 1121 bases of GAP-43 antisense RNA, as described above. Hybridization using 2 × 106 cpm per slide of ³⁵S-labeled antisense or sense riboprobe was performed in a humidified chamber for 5 hours at 50ºC. The tissue sections were then washed in 2× SSC with 10 mM dithiothreitol initially containing 50% formamide, then 50% formamide plus 0.1% Triton-X 100. Single stranded RNA was removed by treatment with 50 μg/ml RNAase A. The sections were further washed in 2× 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. Antibody generation, characterization, and immunohistochemical demonstration of GAP-43

Tissue sections adjacent to those used for in situ hybridization were used for immunohistochemistry. Polyclonal antiserum was raised in rabbits injected with chimeric GAP-43-B-galactosidase fusion protein generated in λgt11 (Karns et al.. Science 236:597-600 (1987); Young et al.. Proc. Natl. Acad. Sci. USA 80:1194-1198 (1983)). Crude serum was processed by ammonium sulfate fractionation and DEAE cellulose chromatography (Horowitz et al., In: Fundamental Techniques in Virology, pp. 297-315). The antibody was assayed by Western blot analysis (Meiri et al.. Proc. Natl. Acad. Sci. USA 83:3537-3541 (1986)) using growth cone membrane particles prepared from neonatal rat brain (Pfenninger et al.. Cell. 35:578-584 (1983)). 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. Neurosci. 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.

RESULTS GAP-43 expression during development assessed by in situ hybridization

At embryonic days 12 and 15, GAP-43 mRNA expression in the CNS was low, but neurons of the dorsal root ganglia exhibited intense labeling, corresponding with their peak period of axonal growth. At E20, GAP-43 levels were uniformly and strikingly high throughout the brain. During the first week of postnatal life, high-level expression persisted, but in contrast to the diffuse labeling observed at E20 and P1, in brains of P7 rats, discrete labeling over individual neurons could be appreciated due to expansion of the neuropil and growth of glial elements. As best as could be determined, all neurons were labeled at this age. At P14, GAP-43 mRNA expression was diminished such that, the overall signal intensity was lower, and only 50-75% of cortical neurons were labeled.

In the adult CNS, 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 P14 brains. The entorhinal cortex, however, 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 ol factory bul b. The pattern of l abel i ng 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.

Visualization of GAP-43 expression by immunohistochemistry The rabbit polyclonal antibody to chimeric GAP-43-B-galactosidase fusion protein generated in λgtll specifically labeled GAP-43 on Western blots of neonatal rat brain. Specific immunostaining for GAP-43 antigen was detected in neurons but not in glial cells. Throughout development, immunoreactive GAP-43 was present in both perikarya and neurites. Neurite-restricted immunoreactive GAP-43 has been observed previously (Benowitz et al .. J. Neurosci. 8:339-352 (1988); Gispen et al., Brain Res. 328:381-385 (1985); Skene et al., Science 233:783-786 (1986)). Cellular fractionation studies suggest that GAP-43 is also in cell bodies (Alexander et aU. J. Biol. Chem. 262:6108-6113 (1987)).

Immunostaining with our antibody permitted localization of GAP-43-expressing cells, and comparison with the in situ hybridization data. The regional distribution and density of neurons containing immunoreactive GAP-43 mirrored the developmental pattern observed for its mRNA by in situ hybridization. GAP-43 immunolabeling was not detected in E12 embryos, and was present in only small amounts (manifested by faint immunohistochemical staining) in the E15 CNS. At E18, GAP-43 immunoreactivity was more conspicuous, and at E20 it was detected at high levels in both somata and neurites of neural cells. This degree of immunostaining for GAP-43 protein persisted through P7. Subsequently, GAP-43 immunoreactivity diminished in most areas, thereby leaving a more restricted distribution of GAP-43 immunoreactivity. In adults, 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.

For example, intense labeling was evident in the hippocampus, in both pyramidal and granule cells throughout CAT, CA4, and the dentate. No labeling occurred when the antibody was preabsorbed with gel-purified GAP-43, isolated as described herein. β-galactosidase preabsorption did not affect the labeling. There were no or few immuno!abeled cells in the cerebellum, brainstem, and spinal cord, low densities of immuno!abeled 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 CA1, CA3 and the dentate gyrus 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

The present example demonstrates that 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.

A recent report by Rosenthal et al.. EMBO J. 6:3641-3646 (1987) also notes inhomogeneous GAP-43 labeling, but somewhat different from that reported here was their finding of higher levels in the cerebellum and frontal cortex, and lack of labeling in the dentate. In previous reports, immunoreactive GAP-43 was detected exclusively in neurites without indication of its cellular origin (Benowitz et al., J. Neurosci. 8:339-352 (1988); Gispen et al., Brain Res. 328:381-385 (1985); Skene et al.. Science 233:783-786 (1986)).

The antibody generated by the present inventors to the β-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.

In any case, 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. It remains to be determined whether the subsets of CNS neurons which persistently express high levels of GAP-43 in the adult share biological features.

One possibility is that 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 315:404-406 (1985)). In fact, there is evidence that such neuronal remodeling is integral to long-term learning (Chang et al.. Brain Res. 309:35-46 (1984); Goelet et al.. Nature 322:419-422 (1986); Horn et al .. J. Neurosci. 5:3161-3168 (1985)) and sexually dimorphic behavior (Kurz et al .. Science 232:395-398 (1986)).

The complement of proteins in growth cones and synaptosomes are not qualitatively very different (Ellis et al., J. Neurosci. 5:1393-1401 (1985); Katz et al.. J. Neurosci. 5:1402-1411 (1985); Sonderegger et al.. Science 221:1294-1297 (1983)) and the growth cone bears markers of its synapses-to- be (Hume et al.. Nature 305:632-634 (1983); Sun et al.. Proc. Natl. Acad. Sci. USA. 84:2540-2544 (1987)). Mature neurons regulate their architecture in part by changing the constituency of molecules transported to their processes (Grafstein et al.. Phvsiol. Rev. 60:1167-1283 (1980); McQuarrie et al.. J. Neurosci. 6:1593-1605 (1986)). Such changes may be mediated locally (Lasek et al., In: Cell Motilitv. RD Goldman. T Pollard. J Rosenbaum. Eds.. Cold Spring Harbor Conferences on Cell Proliferation Series.: Vol 3, Cold Spring Harbor, N.Y., p. 1021-1049 (1976)) and at the level of gene expression, for example, as shown for the tubulins (Miller et al.. J. Cell. Biol. 105:3065-3073 (1987)) and as shown by the present inventors for GAP-43. The characteristics of adult CNS neurons that manifest plasticity are not known, but given the analogies between growth of neurites during development and remodeling of synapses in the mature nervous system, at a molecular level a need for growthrelated proteins is not unreasonable.

Although there is no independent marker to confirm the linkage of GAP-43 to plasticity, there is evidence that some of the neurons which exp.ress high levels of GAP-43 in the adult are capable of synaptic remodeling. Thus, 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. Sorinqer-Verlaq 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. Neurosci. 8:339-352 (1988); Cotman et al.. Psycho! . 33:371-401 (1982); Lee et al.. In: G.A. Kerkut and H.V. Whea! (Eds). Electrophvsiologv of Isolated Mammalian CNS Preparations, Academic, New York, 1981, pp. 189-212; Lee et al.. J. Neurophvsiol. 41:247-258 (1980)) and morphological analysis has confirmed changes in the number and shape of synapses accompanying long-term potentiation (Chang et al., Brain Res. 309:35-46 (1984)). Thus, the restricted localization of GAP-43 in the adult CNS would be compatible with the notion that GAP-43-expressing neurons are those actively engaged in nerve terminal remodeling.

EXAMPLE IV

Dual Regulation of GAP-43 Gene Expression bv Nerve

Growth Factor and Glucocorticoids

In many instances, 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. 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)).

One unifying theme for these two types of plasticity is a structural remodeling, dramatic in establishment of the original and ornate neuronal shape and more subtle in the rearrangement of connections. One notion is that the expression of a set of genes is responsible for neuronal plasticity. Regulation of these genes by the microenvironment would then mediate structural changes.

Corticosteroids are necessary for normal development of the mammalian nervous system, influencing cell fate and neuronal structure and integrity (Doupe et al.. J. Neurosci. 5:2119-2142 (1985); Doupe et al., J. Neurosci. 5:2143-2160 (1985); Anderson and Axel, Cell 47:1079-1090 (1986); Bohn and Lauder, Dev. Neurosci. 1:250-266 (1978); Scheff et al.. Exot. Neurology 68:195-201 (1980); Scheff and Cotman, Expt. Neurology 76:644-654 (1982); Sapolsky et al.. J. Neurosci. 5:1222-1227 (1985)). In culture, cells of neural crest lineage, including small intensely fluorescent (SIF) cells and adrenal medullary cells, may exhibit either neuronal or chromaffin phenotypes. Corticosteroids cause them to assume chromaffin characteristics. In the absence of corticosteroids, the presence of NGF causes them to develop neuronal properties (Doupe et al.. J. Neurosci. 5:2119-2142 (1985)).

Cells of the clonal line PC12, which is derived from a rat adrenal medullary pheochromocytoma (Greene and Tischler, Proc. Nat'l Acad. Sci. USA 73:2424-2428 (1986); Greene and Tischler, in Advances in Cellular Neurobiol ogy. Vol. 3, pp. 373-413, Academic Press, New York (1982)), display a similar bipotential fate, becoming more neuronal with NGF and retaining chromaffin characteristics with exposure to corticosteroids.

Surprisingly, the present investigation of GAP-43 expression has revealed that corticosteroids are powerful negative regulators of GAP-43 gene expression in both PC12 cells and cultured sympathetic neurons. Further, it has been discovered that corticosteroids inhibit the stimulatory effect of NGF on GAP-43 expression.

EXPERIMENTAL PROCEDURES

Materials

Enzymes were purchased from Boehringer Mannheim, New England Biol abs, or Bethesda Research Laboratories, and used as specified by the supplier. Tissue culture products were bought from Gibco. Radiochemicals 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 37ºC. 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. For steroid experiments, 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.

RNA Blotting

Total RNA was prepared from cultured cells by the guanidine isothiocyanate method (Chirgwin et al., Biochemistry 18:5294-5299 (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, 5×SSC (1×SSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0), 1× Denhardt's solution, 1% sodium dodecyl sulfate (SDS) and 100 μg/ml denatured salmon sperm DNA at 42°C.

Hybridization was performed for 10-12 hours at 42°C in the same solution, containing 1x105 cpm/ml of ³²P-labeled DNA probe prepared by random hexanucleotide priming using the Klenow fragment of DNA polymerase I (Feinberg and Vogel stein, Anal. Biochem. 132:6-12 (1984)). The probes were made from cloned cDNAs for GAP-43 as described herein or for GAPDH (Piechaczyk et al.. Nucl. Acids Res. 12:6951-6963 (1984)). Blots were washed with 2×SSC at 65°C twice, each for twenty minutes, and 0.2×SSC for an additional twenty minutes at 65ºC. Autoradiography was performed with intensifying screens at -70ºC. Blots were stripped of hybridized probe at 80ºC for 2 hours in a solution containing lx Denhardt's solution, 1% SDS, 50 mM tris, pH 7.4, and 0.05% sodium pyrophosphate.

Scanning laser densitometry was performed on an LKB ultra scan. Several exposures of a given blot were scanned and the image intensity plotted versus time. Measurements of image intensity were taken from the linear portion of the curve. Careful prior assessment of GAPDH RNA levels showed them not to change under any of these experimental conditions.

Nuclear Run-On Assay

PC12 cultures were split 48-60 hours prior to each experiment. Cells were either left untreated or treated with either NGF (50 ng/ml) or dexamethasone (1 μM) for 6 hours. Approximately ten million nuclei were prepared from each by the method of Greenberg (Greenberg et al.. J. Biol. Chem. 200:14101-14110 (1985)) with the following modifications. Lysis was in 10 mM sodium chloride, 10 mM Tris, pH 7.4, 3 mM calcium chloride and 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 ethyl enediamine tetraacetic acid (EDTA), 2mM dithiothreitol and 200 units/ml RNAsin. Nuclei were counted and stored in liquid nitrogen at a concentration of 50 million/ml.

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 μCi of (³²P) uridi.ne triphosphate were then added (3000 Ci/mmol), and the nuclei labeled for 30 minutes at 30ºC. Nuclei were then digested with 100 units RQ1 DNAase (Promega Biotec) added in 600 μl 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 ºC. The labeled RNA was then digested with proteinase K (Boehringer Mannheim) as described (Greenberg et al.. J. Biol. Chem. 260:14101-14110 (1985)).

After several rounds of phenol, chloroform-isoamyl alcohol extraction, the 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 μl 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 37ºC, and then proteinase K (by adding 100 μl 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 42ºC. After several cycles of phenol, chloroform-isoamyl alcohol extraction, the labeled 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)), glyceraIdehyde-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 μg/ml) was denatured by alkali (0.5 N sodium hydroxide) and neutralized by the addition of ten volumes of 1 M ammonium acetate. Nitrocellulose filter circles were loaded with 50 μg of DNA by gravity filtration. 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 μg/ml of tRNA at 45ºC for 10-12 hours.

Hybridization was performed in the same buffer containing labeled RNA at specific activities ranging from 1-3 × 106 cpm/ml for 4 days at 45ºC. 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 mil l ion hybridized after subtracting background from vector containing filters.

RESULTS Dual regulation of GAP-43 expression

The effects of 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, as determined by densitometry and corrected for RNA loading, 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.

To test the specificity of the steroid effect on accumulated GAP-43 mRNA levels, different steroids of several structural classes were examined over a range of concentrations. Each class of steroid has been shown to selectively affect different types of neurons in vivo (McEwen et al.. Physiological Rev. 66:1121-1188 (1986)).

In one experiment, steroid concentration was 1 μM 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, corticosterone, 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., Science 237:268-275 (1987); Giguere et al.. Cell 46:645-652 (1986)).

Corticosteroids block the NGF induction of a neuronal phenotype in both SIF and adrenal medullary cells (Doupe et al.. J. Neurosci. 5:2119-2142 (1985); Doupe et al., J. Neurosci . 5:2143-2160 (1985)). To investigate the nature of the interaction of the two agents upon GAP-43 expression, PC12 cells were grown for 36 hours in the presence of NGF (50 ng/ml) and dexamethasone (1 μM). The results indicate that dexamethasone prevents the NGF-mediated increase in GAP-43 mRNA.

NGF and steroid effects are direct

The question whether the effects of NGF and corticosteroids are exerted directly or indirectly was addressed by use of the protein synthesis inhibitor cycloheximide. A concentration of 0.5 μg/ml of cycloheximide inhibits more than 94% of (³H) leucine incorporation into protein without an effect on cell viability at 24 hours. Cycloheximide prevents neither the NGF enhancement nor the dexamethasone suppression of GAP-43 gene expression, indicating that neither effect requires de novo protein synthesis. Controls were compared with NGF-treated cells and with cells treated with NGF and 0.5 μg/ml or 2 μg/ml cycloheximide. Larger sized transcripts were noted after treatment with 0.5 μg/ml cycloheximide, which may represent unspliced precursors. Dexamethasone suppression of GAP-43 expression was more pronounced with cycloheximide and dexamethasone than with dexamethasone alone.

These experiments were repeated with the more potent protein synthesis inhibitor anisomycin at a concentration of 0.1 mM for 6 hours, an exposure period used because cell death occurred by 12 hours. Anisomycin did not affect the NGF-mediated increase in GAP-43 mRNA. Dexamethasone suppression was slower than NGF induction, and not discernible by 6 hours, so the effect could not be assayed for anisomycin sensitivity. Pre-treatment with NGF did not prevent direct steroid repression.

In separate experiments, the time required to achieve the fully suppressed steady state levels of GAP-43 mRNA was found to be longer when the steroid was added after NGF pretreatment. Cycloheximide was shown to have no effect on basal GAP-43 mRNA levels.

Steroid repression is transcriptional

To assess the level of regulation involved in both the NGF and steroid effect, nuclear run-on experiments were performed. Nuclei were prepared from PC12 cells treated with 50 ng/ml NGF or 1 μM dexamethasone for 6 hours. The labeled RNA from each group was hybridized to nitrocellulose filters containing immobilized DNAs. After hybridization and washing, the specific radioactivity for each filter was calculated and the data expressed as counts hybridized in parts per million. Dexamethasone decreased the rate of transcription of GAP-43 approximately 4.5 fold, whereas NGF had no appreciable effect on the basal rate of transcription. By comparison, tyrosine hydroxylase transcription increased with dexamethasone and that of GAPDH did not change.

In a series of experiments to define the GAP-43 transcription unit, runoff-labeled RNA prepared from newborn brain nuclei was hybridized to a series of contiguous single strand M13 clones spanning the 5' flanking region through the beginning of the first intron. The results indicated that transcription is from the coding strand only and not from the flanking segment.

Corticosteroids suppress GAP-43 in sympathetic neurons

Although NGF-treated PCI2 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. To do so, dissociated neurons of the rat superior cervical ganglion were cultured, to which 1 μM 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 halflife. DISCUSSION

In this example it is demonstrated that 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. Interestingly and surprisingly, 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.

In vivo, 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. 260:14101-14110 (1985); Milbrandt, Science 238:797-799 (1987); Milbrandt, Neuron 1:183-188 (1988); Leonard et al.. J. Cell. Biol. 106:181-193 (1988)).

The present data indicate that there are several notable differences between GAP-43 regulation and that of these other genes. For some, such as c-fos and NGFIA and NGFIB, NGF induction is rapid, exerted within minutes, and declining after several hours. This is in contrast to the NGF effect on GAP-43 expression, which is slower in onset and persistent.

Additionally, a wide range of stimuli can cause an increase in c-fos, including calcium entry, other growth factors and serum withdrawal and repletion, and these effects are seen in a variety of cell types (Greenberg and Ziff, Nature 311:433-438 (1984)). Genes such as c-fos have been likened to the immediate-early genes of DNA viruses (Goelet et al.. Nature 322:419-422 (1986)), some of which themselves encode transcriptional regulators that reprogram the cellular machinery to a dedicated function.

The delayed response of GAP-43 to NGF suggests that it may fall into a different class of NGF-regulated genes than do c-fos, NGFIA, NGFIB, etc., and may play a role in longer-term adaptation rather than in immediate responses. Unlike these other genes, although NGF does cause a large increase in GAP-43 mRNA accumulation, its effect upon transcription rate is negligible. It is therefore likely that its action is mediated through a post-transcriptional mechanism.

The effects of corticosteroids upon the nervous system are widespread (for review see McEwen et al .. Physiological Rev. 66:1121-1188 (1986)). Since 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.

The present inventors have shown that 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 (Douoe et al.. J. Neurosci. 5:2119-2142 (1985)). Thus, 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 fate. It is of interest that another neural -specific gene, designated SCG 10, is bimodally regulated in PC12 cells (Stein et al.. Develop. Biol. 127:316-325 (1988)). Like GAP-43, SCG 10 gene expression is stimulated by NGF and repressed by glucocorticoids, although the levels of control differ somewhat between the two genes.

Corticosteroids do not noticeably affect PC12 cell shape. Since GAP-43 is suppressed, it is clear that normal levels of mRNA are not needed for neurite extension in PC12 cells. However, it is not clear that these results may be interpreted to mean that GAP-43 is unnecessary for neurite growth. First, low levels of GAP-43 RNA are still present after steroidsuppression. Second, 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.

EXAMPLE V

The Neuronal Growth-Associated Protein GAP-43 Imparts

Growth Cone-Like Morphology to Non-Neuronal Cells In the present example, the inventors set out to test the hypothesis that GAP-43 might contribute directly to the establishment of the unique neuronal phenotype, perhaps at the level of the cytostructure. Thus, in yet another aspect of the invention, 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, L.R. et al.. Science 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, Y., Cell 23:175 (1981)) were transfected as described (Zuber, M.X. et al., Science 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, B. et al .. Proc. Natl. Acad. Sci. 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. In GAP-43 transfected cells, GAP-43 immunoreactivity was prominent in about 5-20% of the cells, depending upon transfection efficiency. Some GAP-43 appeared to be membrane-associated, an observation confirmed by Western blot analysis comparing cytosolic and membrane fractions.

Cells that expressed high levels of GAP-43 had a distinctive structure with many cells extending processes from their cell perimeter. To ensure that this was not due to better visualization of the cell surface by antibody to GAP-43, the surface of all cells was labeled by rhodaminated wheat germ agglutinin. CD8-transfected cells, mock transfected cells, or cells in the same GAP-43 transfected dish that did not express GAP-43, did not have these extensive processes, although shorter or single processes did occur. Long, thin processes appeared to be associated only with high level GAP-43 expression. A similar association of process outgrowth with high level GAP-43 expression was found when WOP cells, 3T3 cells expressing polyoma T antigen (Valle et al.. Mol. Cell. Biol. 1:417 (1981)), were transfected with CDM8-GAP, a GAP-43 expression vector that included the polyoma origin of replication. In these transient transfection assays, the efficiency of transfection and level of expression both vary, making difficult the quantitation of the effect.

To overcome the problem of quantitation, a series of clonal, stably transformed CHO cell lines was generated that constitutively expressed GAP-43. Control cell lines 30 minutes after plating were generally round. In GAP-43- expressing lines, GAP-43 expression clearly correlated with process extension. Many cells expressing GAP-43 extended filopodial processes that were narrow and between 20 and 75 μm in length. In both control and cell lines expressing GAP-43, the perimeter often included broad, thin, ruffled lamellipodia.

Clonal cell lines constitutively expressing GAP-43 were established by co-transfection of CDM8-GAP and Neomycin resistance expression plasmids into CHO cells by the Ca-PO4 co-precipitation method and G418 selection (Maniatis, T. et al .. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982)). In control transfected cells, the plasmid pCDM8 (Seed, B., Nature 329:840 (1987)) was used instead of CDM8-GAP. After the cells became confluent, they were passaged with trypsin and plated on polyD-lysine-coated glass coverslips.

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.

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. However, there is evidence that GAP-43 is, in fact, related to growth cone function, in that it is enriched in growth cones (Katz, F. et al.. J. Neurosci . £:1402 (1985); DeGraan, P.N.E. et al.. Neurosci .61:235 (1985); Meiri, K.F. et al.. Proc. Natl. Acad. Sci. USA 83:3537 (1986); Skene, J.H.P. et al.. Science 233:738 (1986)), is at its highest levels in neurons extending axons in vivo or in vitro (Skene, J.H.P. et al.. J. Cell Biol. 89:86 (1981); Benowitz, V.E. et al.. Neurosci. 1:300 (1981); Meiri, K.F. et al., J. Neurosci. 8:2571; Benowitz, L.I. et al., T.I.N.S. 10:527 (1987)), and increases in PC12 cells with NGF exposure concomitant with neurite growth, as described above.

Although the inventors do not intend to be bound by a particular theory, one interpretation of these data is that 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, D. et al .. Science 239:883 (1988); Smith, S.J., Science 242:708 (1988)).

Many cells can extend filopodia or lamellipodia, a tendency that depends upon several factors, including the phase of cell cycle, plating conditions, and levels of second messengers (Allred, L.E. et al .. Surfaces of Normal and Malignant Cells R.O. Hynes, Ed. (John Wiley & Sons, New York, 1979), p. 21). For this reason, cells were assayed under exactly the same strict plating conditions. The cellular mechanisms that permit cells to extend processes are complex, and include components which are likely to be present in all cells. Therefore, the similarity of fibroblast processes to those of growth cones is not surprising. In fact, 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, D. et al.. Science 239:883 (1988); Smith, S.J., Science 242:708 (1988)). How GAP-43 might interact with such machinery remains to be determined.

EXAMPLE VI

Identification of a Novel Membrane-Targeting Peptide

In yet another aspect of the present invention, it has been found that the GAP-43 protein contains a novel membranetargeting 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 membranetargeting 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.

According to the 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.

It is well known that most membrane-associated proteins contain a highly hydrophobic domain which directly intercalates with the cell membrane. Surprisingly, however, it has been discovered that 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 surprisingly 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 carboxyterminus of GAP-43 had no effect on membrane binding. However, a synthetic GAP-43 gene lacking the initial four amino acids (MET LEU CYS CYS), and beginning at the MET of position five failed to bind to the membranes of neuronal or non-neuronal host cells (see Figure 11), indicating that the first exon is responsible for this membrane-targeting function.

By "membrane-targeting peptide," then, is meant any amino acid sequence as follows:

MET LEU CYS CYS MET ARG ARG THR LYS GLN

or a functional derivative thereof, which, when attached at or near the amino-terminus end of a desired protein or peptide, will effect the direction of said protein or peptide to the cell membrane.

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.

Thus, in another aspect, the present invention provides for cDNA coding for a membrane-targeting domain comprising the nucleotide sequence

atg ctg tgc tgt atg aga aga ace aaa cag or a functional derivative thereof. Because of the degeneracy of the genetic code, of course, it will be possible to vary the nucleotides while still achieving the desired results. Similarly, those of skill will appreciate that, in certain instances, it may be desirable to alter the nucleotides when expression is contemplated in a particular host, because of preferred codon usage. These and other such modifications are contemplated as within the scope of the present invention.

In order to further elucidate the GAP-43 membranetargeting domain, non-neuronal cells (including COS cells, NIH 3T3 cells, and CHO cells) and neuronal cells (PC12 cells) were transfected with 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).

It was found that mutation of either C3 or C4 results in a significant reduction in membrane binding. The most marked effect was seen when C4 was altered. Mutation of both C3 and C4 completely abrogated the phenomenon. The mechanism of this effect is unclear, but is apparently unrelated to simple alterations in oxidation state at those positions, since redox experiments failed to alter membrane binding.

Further, a synthetic GAP-43 gene was constructed which lacked the initial four amino acids (MET LEU CYS CYS), and began at the MET of position five. The expressed protein failed to bind to the membranes of neuronal or non-neuronal host cells. (See Figure 11.) This shows that the first four amino acids are necessary for membrane binding. As discussed below, experiments with normally cytosolic proteins have demonstrated that the ten amino acid peptide is clearly sufficient. Preliminary data indicate that the first four amino acids are also sufficient for membrane binding. Thus, in another embodiment, the invention comprises an amino acid sequence comprising

MET LEU CYS CYS

or a functional derivative thereof, which sequence may be attached at the amino-terminus end of a desired protein or peptide in order to allow membrane binding of said protein or peptide.

Moreover, 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.

In an additional experiment, 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 membraneassociated. Plasmids containing this sequence were used to transfect neuronal and non-neuronal cells (Figure 11). Immunofluorescence assay revealed that the expressed CAT protein was membrane-associated in transfected cells. This demonstrates that the amino acids of the first GAP-43 exon are sufficient to accomplish membrane targeting of a desired protein or peptide.

Moreover, experiments have shown that the first 40 amino acids of 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.

To further elucidate the selective growth cone accumulation phenomenon described herein, 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. Constructions that encoded varying amounts of the GAP-43 amino terminus fused to a reporter peptide were expressed in COS and PC12 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) .

Immunoblotting was carried out as follows: Chimeric proteins with the amino terminus of GAP-43 fused to CAT associate with COS cell membranes. CAT, GAP10CAT and GAP40CAT were transiently expressed in COS cells. Immunoblots of membrane (M) and cytosolic (C) fractions from each transfection were prepared using anti-CAT antibody. In the CAT-transfected cells, immunoreactivity is found only in the cytosolic fraction and co-migrates with purified CAT protein. In the GAP40CAT and GAP10CAT 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 PC12 cells over-expressing GAP-43, nearly all of the GAP-immunoreactivity is membrane-associated and co-migrates with purified GAP-43.

In the GAP-43 expression plasmid, pGAP, the GAP-43 coding sequence replaced the stuffer at the Xba I sites of the CDM8 plasmid described by Seed, B. 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 3AI site 68 bp downstream from the termination codon, as described herein. For the CAT expression plasmid, pCAT, the Hind III to Bam HI fragment containing the CAT coding sequence and polyadenylation site from pSV2CAT (Gorman, CM., Moffat, L.F. & Howard, B.H. Mol. Cell. Biol. 2:1044-1051 (1982)) replaced the Hind III to Bam HI fragment of CDM8 containing the stuffer and polyadenylation site.

pGAP40CAT and pGAP10CAT include the first forty or ten amino acids of GAP-43, respectively, fused in-frame with CAT in pCAT by the use of poly! inkers. For transient transfection of COS cells, DEAE dextran and chloroquine was used as described (Zuber, M.X., Simpson, E.R. & Waterman, M.R. Science 234:1258-1261 (1986)). 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 PC12 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-lysine-coated covers! ips in the presence of 50 ng/ml NGF and analyzed 24 hours later.

For immunochemical assays, 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.

For cell fractionation, COS or PC12 cells were scraped from 100 mm confluent petri dishes and pelleted at 2000 × g for 10 minutes. The pelleted cells were homogenized by Polytron in 10 mM Tris- HCl, 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. (28) and used as a positive control for immunostaining. The same volume of cytosol or membrane fraction (usually 100 ul) was electrophoresed on polyacryl amide gels (29). Proteins were electrophoretically transferred to nitrocellulose and excess sites were blocked with 4% BSA. Membranes were then incubated for 24 hour at 4ºC 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 (Kirkegaard and Perry, Gaithersburg, MD) was employed as peroxidase substrate.

Immunoblotting revealed that CAT expressed in COS cells or PC12 cells is present only in the cytosolic fraction. By contrast, the chimeric proteins GAP10CAT and GAP40CAT are membrane-associated. The fusion protein is extracted by detergent, but not by sodium chloride, calcium chloride, or EGTA. Thus, the nature of this membrane binding is similar to that of native GAP-43 in rat brain (Perrone-Bizzozero, N.I., Weiner, D., Hauser, G. & Benowitz, L.I. J. Neurosci. Res. 20:346-350 (1988); Oestreicher, A.B., Van Dongen, C.J., Zwiers, H. & Gispen, W.H. J. Neurochem. 41:331-340 (1983); Chan, S.Y., Murakami, K. & Routtenberg, A. J. Neurosci. 6:3618-3627 (1986); Skene, J.H.P. & Virag, I. J. Cell Biol. 108:613-624 (1989).

The cellular distribution of 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. By this assay, 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, C.O.M., Holthuis, CM., Oestreicher, A.B., Boonstra, J., De Graan, P.N.E. & Gispen, W.H. J. Cell Biol. 108:1115-1125 (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) GAP10CAT 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 GAP10CAT, whereas anti -GAP-43 antibody was used for GAP-43. Control PC12 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. The samples were blocked with 4% BSA in PBS for 1 hour, incubated for 1 hour in primary antibody, rinsed with PBS, incubated in 0.3% H₂O₂ in PBS for 15 minutes (to reduce background), rinsed again and incubated 1 hour in secondary antibody. After washing with PBS several times, coverslips were rinsed with water and mounted with Gelvatol containing 0.4% n-propyl gall ate to decrease bleaching. Immunofluorescence was not detectable above background when cells did not contain specific antigens or when the primary or secondary antibodies were omitted.

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, K., Schreyer, D.J., Skene, J.H.P. & Banker, G. 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.

For high resolution confocal microscopy, the cells were fixed with freshly made 4% paraformaldehyde and 0.5% glutaraldehyde, which was essential to preserve the fine structure of the filopodia, followed by 0.1% Triton-x-100 for 3 minutes and 10 minutes with 2 mg/ml sodium borohydrate in PBS. Confocal analysis employed a Biorad MRC-500 scanning confocal imaging system and a Zeiss Axioplan microscope.

These experiments confirm the present inventors surprising discovery that the first 10 amino acids of GAP-43 suffice to direct growth cone accumulation. The present inventors are not aware of other proteins that have a sequence closely related to the GAP-43 amino terminus, although at least one other non-integral membrane protein that accumulates in growth cone membranes, SCG 10 (Stein, R., Mori, N., Matthews, K., Lo, L.-C & Anderson, D.J. Neuron positions 22 and 24).

In pol arized epithel i al cel l s , different proteins accumul ate in the apical and basol ateral pl asma membranes (Mat! i n , K.S. J . Cell Biol . 103 : 2565-2568 (1986) ; Rodriguez-Boulan, E.J. & Sabatini, D.D. Proc. Natl. Acad. Sci. USA 75:5071-5075 (1978); Simmons, K. & Fuller, S.D. Ann Rev. Cell Biol. 1:243-288 (1985)) a process believed to depend upon sorting signals within the protein, similar to the signals which direct traffic of membrane and secreted proteins to their particular destinations (Wickner, W.T. & Lodish, H.F. Science 230:400-407 (1985); Verner, K. & Schatz, G. Science 241:1307-1313 (1988); Pfeffer, S.R. & Rothman, J.E. Ann. Rev. Biochem. 56:829-852 (1987)). In the case of epithelial cells, such signals would also recognize different regions of the plasma membrane as apical or basolateral.

In neurons, 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. While the present inventors do not intend to be bound by any particular theory, it seems less likely that the palmitylated residues interact with the lipid bilayer directly, because that would likely cause a more uniform membrane distribution for GAP-43. Along these lines, the fatty acid moiety of another acylated protein, N-myristylated VP4 of poliovirus, has been shown by X-ray diffraction to interact with specific amino acid residues of other viral proteins and not with the lipid bilayer (Schultz, A.M., Henderson, L.E. & Oroszlan, S. Ann. Rev. Cell Biol. 4:611-647 (1988); Chow, M., Newman, J.F., Filman, D., Hogle, J.M., Rowlands, D.J. & Brown, F. Nature 327:482-486 (1987)). Since 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.

It is notable that the sorting domain of 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, C.O.M., Holthuis, CM., Oestreicher, A.Bo, Boonstra, J., De Graan, P.N.E. & Gispen, W.H. J. Cell Biol. 108:1115-1125 (1989)). Given the observation that transfected GAP-43 enhances the propensity of non-neuronal cells to extend filopodia as described herein, it will be of interest to correlate GAP-43 location with motile activity of particular filopodia.

Thus, 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. In one exemplary embodiment is provided a method for directing a desired protein or peptide to the membrane of a cell, comprising

(a) ligating to the amino-terminus of said protein or peptide a membrane-targeting peptide comprising an amino acid sequence selected from the group consisting of

I. MET LEU CYS CYS MET ARG ARG THR LYS GLN;

II. MET LEU CYS CYS MET ARG ARG THR LYS;

III. MET LEU CYS CYS MET ARG ARG THR;

IV. MET LEU CYS CYS MET ARG ARG;

V. MET LEU CYS CYS MET ARG;

VI. MET LEU CYS CYS MET;

VII. MET LEU CYS CYS; and

VIII. functional derivatives thereof; and

(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 membranetargeting domain.

In another non-limiting exemplary embodiment, the present invention provides nucleotide sequences encoding the membranetargeting 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 of eukaryotic hosts by methods well known to those of skill.

As described herein, of course, 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. EXAMPLE VII

Cloning of the Entire Rat Genomic DNA of GAP-43 and Identification of a Regulatory Site The work of the present inventors as described herein strongly suggests that 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. It has surprisingly been discovered that 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.

Further, 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.

These results suggest that brain-specific nuclear proteins bind to a specific region upstream of GAP-43. EXPERIMENTAL PROCEDURES

Genomic cloning and mapping

All methods used for cloning were as described by Ausubel et al . , Eds., Current Protocols in Molecular Biology. John Wiley & Sons, publisher (1987). All enzymes were purchased from New England Biolabs. Genomic clones containing the three GAP-43 exons were isolated from a library constructed by inserting size fractioned SauIIIA 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. To find exon I, 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.

H-DNA gels

Two 25 cm long, 1.4% agarose gels were poured using 45 mM Tris base (adjusted to pH 7.4 or pH 4.0 with acetic acid) as a buffer. Loading buffer was electrophoresis buffer containing 5% glycerol, 0.1% bromophenol blue and 0.1% xylene cyanol. Gels were loaded with the digests of exon 1 containing plasmid bsl.5R1X4 listed in the legend of Figure 15, run for 16 hr at 20V, stained with ethidium bromide in Tris acetate pH 9, destained and photographed.

Sequencing

The GAP-43 promoter was sequenced by the dideoxy method of Sanger et al .. Proc. Natl. Acad. Sci. 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, Meth. Enzvmol. 101:20-78 (1983)) for single-stranded sequencing.

RNAse mapping

The RNAse protection analysis was done as described in Krieg and Melton, Meth. Enzvmol. 155:397-415 (1987). For the protections, 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.

Other 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 PC12 cells.

In another RNAse protection analysis, 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. In all cases, transcripts were elongated with IT 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. RESULTS

Cloning of GAP-43 genomic sequences

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 our 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 representations 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 calmodul in 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 intron-exon boundaries shown in Figure 13b were identified by sequencing and are in agreement with consensus splice sites (Mount, Nucl. Acids Res. 10:459-472 (1982)). The polyadenylation site shown here was verified by RNAse protection and agrees with the one predicted by Rosenthal et al.. EMBO 6:3641-3646 (1987) as the major site. A tandem pair of the consensus motif (YGTGTTYY) often found immediately 3' of poly A addition sites (McLauchlan, Nucl. Acids Res. 13:1347-1368 (1985)) is underlined in the figure.

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 55:505-518 (1988); Ingraham et al.. Cell 55:519-529 (1988)).

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). Hairpin forming palindromes centered at -112, -232 and -509 flank the homopolymer regions and may influence secondary structure.

Purine-pyrimidine homopolymer stretches, especially those with mirror symmetry (Mirkin, Nature 330:495-497 (1987)), have the potential to assume a triple stranded conformation termed H-DNA (for reviews see Wells et al.. J. Biol. Chem. 263:1095-- 1098 (1988); Htun and Dahlberg, Science 243:1571-1576 (1989)).

The first indication that the GAP-43 promoter contained regions of strong secondary structure in vitro came while sequencing it. Sequencing is routinely accomplished using the double stranded dideoxy method, but when this technique was applied to the GAP-43 promoter, readable sequence would come to an abrupt halt upon reaching the homopolymer region at -250. Only after subcloning small fragments into M13 for single stranded sequencing were the inventors able to arrive at the sequence.

Htun and Dahlberg, Science 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 and Dahlberg, (1988), supra. 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. In carrying out gel electrophoresis, aliquots of the digests represented in Figure 15 were loaded on 1.4% agarose gels that had been equilibrated with Trisacetate at either pH 7.4 or 4.0 and run in parallel. Bands that shifted at pH 4 exhibited smearing that may result from the B to H transition (Htun and Dahlberg, Science 241:1791-1795 (1988)). Only bands containing homopolymer region I (i.e., the fragments containing the upstream (-240 to -370) homopurine stretch) exhibited an altered mobility at pH 4.0 in this assay. There was no visible shift in the markers or in fragments of the plasmid from outside the promoter region, or even in the fragments containing regions II and III when they were separated from region I. Thus, only the upstream region exhibited a shift on its own. Note that the fragments containing regions II and III did not shift at pH 4. Progressive removal of DNA outside the homopolymer region increased the relative shift in mobility. A much greater shift in mobility was observed when region II was included in a fragment with region I than when region I alone was present in a similarly sized fragment. In this assay, regions II and III were not able to effect a shift on their own, but they may act cooperatively with the upstream region.

Heterogeneity of GAP-43 transcription initiation

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 -47/-48, -51/-52, and -78. These same sites were identified by primer extension. Additional minor bands become visible after longer exposure.

Several transcripts at around -230 are present to a much greater extent in mRNA from the cerebral cortex as compared to the dorsal root ganglia. This is interesting in light of observations that suggest the regulation of GAP-43 gene expression in the central and peripheral nervous system is different (Skene et al.. J. Cell Biol. 89:86-95; J. Cell Biol. 89:96-103 (1981)). Hence, RNA from other areas of the CNS, as well as from PC12 cells (which are believed to derive from sympatho-adrenal precursor cells), 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 PC12 cells, although the amount of each of these longer messages is relatively small. When 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. This analysis showed that the longer GAP-43 transcripts together actually account for a significant fraction of the total GAP-43 transcripts, and that these transcripts are much more prevalent in RNA from the CNS than that from the DRG or PC12 cells. In sum, the 5'end of GAP-43 mRNA is heterogeneous, and upstream start sites are used more commonly in the central nervous system as compared to the peripheral nervous system. DISCUSSION

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 J. 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 necessary for membrane binding of GAP-43, and contains sufficient information to target heterologous fusion proteins to the same membrane domains as GAP-43, including those of the growth cone, as described hereinabove.

The second exon includes the calmodul in 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 and Zwiers, Soc. Neurosci. Abstract (1988)). Exons I and II contain regions that are well conserved between fish and several mammalian GAP-43 proteins (Labate and Skene, (1989)).

The promoter of GAP-43 is unusual in sequence and structure. 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)). Furthermore, the tightly regulated expression of GAP-43 in development, its specificity to neurons, and its inducibifity 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 and Willard, J. Cell Biol. 89:96-103 (1981). As described hereinabove, GAP-43 does not appear to be irreversibly repressed in the CNS, and may play a role in plasticity other than in axonal growth (Benowitz and Routtenberg, T.I.N.S. 10:527-532 (1987)), but it is clear that there is a difference in regulation centrally and peripherally. Hence, the usage of different start sites suggests the possibility that the mRNA from different neurons may differ at the 5' end, in turn regulating ribosome binding or stability.

It is interesting to note that 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)). Also, like GAP-43, 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 (ibid.). This suggests an additional level of control in brain versus other tissues for both GAP-43 and Thy-1.

A potential upstream regulatory element present in the GAP-43 promoter is the consensus Pit-1 binding site (TAT-TCATG). This and related sequences are recognized and bound by transcription factors known collectively as POU proteins. This group originally included Pit-1, Oct-1 and Oct-2 in mammals, and unc86 in nematodes (reviewed in Herr et al .. Genes Develop. 2:1513-1516 (1988)), and has recently been expanded by the finding of cDNAs encoding proteins that share the two peptide regions that characterize this family (He et aL., Nature 340:35-42 (1989)). As described in the following example, the present inventors have identified and cloned brain-specific proteins that bind this region of GAP-43 and may regulate its transcription.

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 and Tjian, Cell 53:699-711 (1988); Gilmour et al.. Science 245:1487-1490 (1989)), and because they have the potential to take on a triple stranded conforma tion 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. 2:2939-2949 (1988); Htun and Dahlberg, Science 243:1571-1576 (1989)). For instance, it has been postulated that adoption of the H configuration, perhaps stabilized by protein interactions, would cause a kink in the DNA. This kink could phase nucleosomes by exclusion from the kinked region, thereby making the DNA around the kink more accessible to transcriptional factors (Htun and Dahlberg, Science 241:1791-1795 (1988); Han and Grunstein, Cell 55:1137-1145 (1988)). Additionally, such a kink could serve to bring upstream sequences into closer apposition to those downstream, allowing an interaction between the sequences or proteins bound to them (Htun and Dahlberg, Science 241:1791-1795 (1988)). Alternatively, H-DNA could serve as a repressor of transcription by directly blocking access to DNA in its immediate vicinity (Maher et al.. Science 245:725-730 (1989)).

EXAMPLE VIII

A Major Component of the Neuronal Growth Cone Membrane is the GTP Binding Protein, G₀

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, G₀. Immunohistologic staining of neuronally differentiated rat pheochromocytoma cells demonstrates high concentrations of the alpha subunit of G₀ at the distal tips of cellular processes. These data suggest that regulation of growth cone motility may utilize a G₀ signal transduction mechanism.

The complex state of neuronal connectivity achieved during brain development, and refined through synaptic plasticity, requires selection of specific targets by neuronal axons. The mechanisms by which axons transduce information form their extracellular milieu into directed growth are poorly understood. 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, D., et al ., Ann. Rev. Cell Biol. 4:43 (1988)). Fortunately, the membrane of the axonal growth cone, and therefore its transduction system, can be fractionated from other neuronal constituents (Pfenninger, K.H., et al.. Cell 35:573 (1983); Gordon-Weeks, P., et al.. Neuroscience 13:119 (1984); Ellis, L., et al.. J. Cell Biol. 101:1977 (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, K.H., et al.. Cell 35:573 (1983); Ellis, L., et al.. J. Cell Biol. 101:1977 (1985); Simkowitz, P., et al.. J. Neurosci. 9:1004 (1989); Cheng, N., et al .. J . Biol. Chem. 263:3935 (1988); Meiri, K.F., et al .. Proc. Natl. Acad. Sci. USA 85:3537 (1986); Skene, J.H.P., et al .. Science 233:783 (1986)). Characterization of the other major growth cone membrane constituents could explain axonal response to extracellular cues.

A growth cone membrane fraction was prepared from neonatal rat brain (Pfenninger, K.H., et al .. Cell 35:573 (1983); Ellis, L., et al.. J. Cell Biol. 101:1977 (1985); Simkowitz, P., et al .. J. Neurosci. 9:1004 (1989); Cheng, N., et al.. J. Biol. Chem. 263:3935 (1988)). This preparation has a simple protein composition by SDS-PAGE (Ellis, L., et al .. J. Cell Biol. 101:1977 (1985); Simkowitz, P., et al.. J. Neurosci. 9:1004 (1989); Cheng, N., et al.. J. Biol. Chem. £61:3935 (1988)). The most intensely stained ban, migrating at 50-55,000 daltons, has been identified as tubulin (Simkowitz, P., et al.. J. Neurosci. 9:1004 (1989); Cheng, N., et al., J. Biol. Chem. 263:3935 (1988)). There are also prominent proteins with M_rs of about 35,000 and 40,000, which have been termed p34 and p38, and are specifically enriched in the growth cone membrane (Simkowitz, P., et al., J. Neurosci. 9:1004 (1989)). These are the two unidentified proteins which were characterized further. Apart from cytoskeletal proteins, they are the most prominent proteins in the growth cone membrane preparation.

It was noted that p34 and p38 have similar molecular weights to the alpha and beta subunits of the GTP-binding protein, G₀ (Stryer, L., et al.. Ann. Rev. Cell Biol. 2:391 (1986); Gilman, A.G., Ann. Rev. Biochem. 56:615 (1987)). Coelectrophoresis of the growth cone membranes with purified bovine grain G₀ demonstrated that p34 co-migrates with the beta subunit, and p38 with the alpha subunit of G₀. Immunoblotting demonstrated that p34 reacts with an anti-beta subunit antiserum, and that p38 reacts with an anti-alpha ^• subunit G₀ antiserum. Furthermore, the predominant protein species of p38 must be alpha₀, because equal protein concentrations of p38 and alpha₀, as determined by Coomassie blue staining, exhibited identical immunoreactivity. The same was true for p34 and the beta subunit of G₀. Alpha-_j subunit was about 10-fold less reactive than alpha₀ with this antiserum (Gilman, A.G., Ann. Rev. Biochem. 56:615 (1987)), so that it cannot account for a major percentage of the alpha₀ immunoreactivity.

To verify these immuno!ogic and electrophoretic data, partial protein sequences were obtained from electrophoret ically purified p34 and p38 (Figure 16). Both p34 and p38 were amino termimally blocked. Sequence was obtained from tryptic fragments separated by HPLC and from Staoh. aureus V8 protease partial digestion fragments separated by SDS-PAGE. The sequence for each of three peptides from p38 was identical to that of alpha₀, confirming that alpha₀ is the major component of the p38 protein. Other known alpha subunits have similar but distinct sequences. The three p34 peptides had a sequence identical to that of the beta subunit of G proteins. Two peptides were from regions where beta₁ and beta₂ subunits are identical and the third contained a mixture of the sequences for beta₁ and beta₂. Thus, the alpha and beta subunits of G₀ are major constituents of the growth cone membrane subcellular fraction.

Although these preparations are substantially enriched in growth cone membrane, they are not pure (Pfenninger, K.H., et al.. Cell 35:573 (1983); Gordon-Weeks, P., et al. , Neuroscience 13:119 (1984)). Previous immunohistology of unfractionated tissue has demonstrated that G₀ is concentrated in the neuropil of adult rat brain (Worley, P.F., et al .. Proc. Natl. Acad. Sci. USA 83:4561 (1986)), that a related G protein, G_0lf, is localized to the terminal region of primary olfactory neurons in the adult (Jones, D.T., et al .. Science 244:790 (1989)), and that G₀ stains throughout cultured primary neurons but is concentrated at regions of cell-cell contact (Jones, D.T., et al.. Science 244:790 (1989)). These studies are consistent with, but do not prove, G₀ localization in growth cones. Therefore, immunohistologic methods were employed on NGF-treated PC12 cells to examine G₀ distribution in the intact cells. NGF causes these cells to extend long processes tipped with growth cones. The neuronal protein GAP-43 is enriched in these growth cones as it is in those of primary neurons (Van Hooff, C.O.M., et al ., J. Cell Biol. 108:1115 (1989)). Alpha₀ immunofluorescence was highly concentrated in these growing tips of PC12 cells (although it was not found exclusively there).

METHODS

Preparation of growth cone membranes

Growth cone membranes were prepared with minor modifications from previous methods (Pfenninger, K.H., et al .. Cell 15:573 (1983); Ellis, L., et al.. J. Cell Biol. 101:1977 (1985); Simkowitz, P., et al.. J. Neurosci. 9:1004 (1989); Cheng, N., et al.. J. Biol. Chem. 263:3935 (1988)). SpragueDawley rats less than 24 hours old were decapitated and the brains were homogenized at 4ºC with 6 passes in a glass/teflon homogenizer in 5 volumes of 0.32 M sucrose, 1 mM Tris-HCl, 1 mM MgCl, pH 7.6. The following 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 × g for 40 min. The membranes were washed by resuspension in 20 ug/ml saponin and 0.3 M Na₂So₃ and again collected by centrifugation. Bovine brain G₀ was prepared as described (Bray, D., et al.. Ann. Rev. Cell Biol. 4:43 (1988)).

Production of anti-sera

The production and characterization of anti-bovine brain alpha₀ and anti-beta antiserum in rabbits has been described (Huff, R.M., et al .. J. Biol. Chem. 260:10864 (1985)). Immunoblot samples were electrophoresed through 10% polyacrylamide gels with SDS and then electrophoretically transferred to nitrocellulose. Non-specific protein binding sites were blocked with 10 mg/ml bovine serum albumin, and the blots were incubated with 1:400 anti-alpha antiserum or 1:100 anti-beta antiserum (Huff, R.M., et al., J. Biol. Chem. 260:10864 (1985)) overnight at 4º. Bound antibody was detected by the avidin biotin complex method (Vectastatin, Burlingame, CA) using tetrabenzidine as a peroxidase substrate.

Amino acid sequencing of growth come membrane proteins

Growth cone membranes -were fractionated as described above. When proteins were transferred to polyvinylfluoridine (PDVF) membranes, the spots for p34 and p38 yielded no sequences, presumably because the proteins are amino terminally blocked. Therefore, protein sequences were obtained from proteolytic fragments for p34 and p38. For tryptic digestion, the proteins were transferred to nitrocellulose, stained with ponceau S and the appropriate bands were excised. At the Harvard Microchemistry Facility, tryptic digestions were performed on the nitrocellulose membranes, and the released peptides were separated by reverse phase HPLC and sequenced on a gas phase automated sequenator (Moos, M., et al . J. Biol. Chem. 263:6005 (1988)). Further amino acid sequence was obtained for p34 following partial digestion with Staph. Aureus V8 protease (Boehringer, Mannheim). Polyacryl amide cubes containing p34 were digested in situ with V8, fractionated by SDS-PAGE, electroblotted to PVDF membrane (Millipore, Bedford, MA) and visualized with Coomassie blue (Kennedy, T.E., et al.. Proc. Natl. Acad. Sci. USA 85:7008 (1988)). Peptide fragments were excised and sequenced on an Applied Biosystems (Foster City, CA) 470A gas phase sequencer at the Howard Hughes Medical Institute Protein Chemistry Core Facility of Columbia University to yield sequence p34-B. Alpha₀ immunostaining of PC12 cells

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 alpha₀ antiserum for 1 hour at 23ºC, rinsed with PBS, and incubated with 0.3% H₂O₂ for 15 minutes to reduce background. Bound rabbit immunoglobulin was detected by use of the Texas red conjugated donkey anti-rabbit IgG (Jackson Immunologicals).

RESULTS

SDS-PAGE reveals two bands which co-migrate with G₀ alpha and beta subunits

Proteins of the axonal growth cone membrane were identified by SDS-PAGE. Two separate preparations of axonal growth cone membranes, purified bovine brain G₀, and crude brain homogenate were electrophoresed through a 10% polyacrylamide gel in the presence of SDS and stained with Coomassie blue. Enrichment of two bands, termed p34 and p38, in the growth cone membrane preparation relative to crude brain was observed. These proteins comigrated with the alpha and beta subunits of purified G₀. Under these conditions, actin and GAP-43 comigrated with an apparent Mr of 43,000.

Anti-G₀ immunoblots of growth cone membrane show G0 reactivity Immunoblotting revealed alpha₀ immunoreactivity migrating at the position of alpha Coomassie blue staining in both the purified G₀ preparation and the growth cone membrane preparation. The gels were loaded such that Coomassie blue labeling of alpha₀ was identical to that of P38, and similarly matched for other pairs. The Coomassie stained gels were run 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 alpha₀ with this antiserum. This suggests that most or all of p38 is alpha₀. There was a small amount of immunoreactivity migrating just above alpha₀ in the G₀ sample which was likely to be due to slight contamination and cross-reaction with alphai. This was not seen in the growth and growth cone membrane (A) fractions, which were previously shown to stain identically with Coomassie blue for beta and p34, respectively, and also showed similar immunoreactivity with anti-beta antiserum.

The partial protein sequence for p34 and p38 is identical to that of G₀.

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 alpha₀ from rat brain (Goh, J.W., Science 244:980 (1989)). The sequence of three peptides from p34 is compared to that of betaj and beta₂ subunits from bovine brain (Fong, H.K.W., et al .. Proc. Natl. Acad. Sci. USA 84:3792 (1987)). Note that two peptides are identical to regions in which betaj and beta₂ are identical. The other peptide contains a mixture of the sequences for beta₁ and beta₂.

Alpha₀ immunoreactivity is concentrated in the tips of PC12 processes

Alpha₀ staining of PC12 cells differentiated with nerve growth factor revealed high concentrations of the antigen at the distal tips of the cellular processes. There was also a lower level of diffuse staining in the region of the cell body surrounding the nucleus. The specificity of the antibody has been demonstrated (Huff, R.M., et al.. J. Biol. Chem. 260:10864 (1985); Worley, P.F., et al.. Proc. Natl. Acad. Sci. USA 83:4561 (1986)), but as a further control, identical samples were prepared with the addition of excess purified bovine brain G₀ (30 ug/ml) to the incubation with antiserum, or with the substitution of normal rabbit serum for antiserum. These controls exhibited essentially no staining of the cellular processes.

DISCUSSION

The results presented in the present example demonstrate that a G protein, specifically G₀, is a major constituent of the growth cone membrane. In fact, there is more G₀ than any other non-cytoskeletal protein in the growth cone membrane. G proteins, in general, couple transmembrane receptors to intracellular signalling systems, although the role of G₀, which is expressed primarily in brain, has not been clear (Stryer, L., et al., Ann. Rev. Cell Biol. 2:391 (1986); Gilman, A.G., Ann. Rev. Biochem. 56:615 (1987); Neer, E.J., Nature 333:129 (1988); Ross, E.M., Neuron 3:141 (1989)). G₀ can interact with a number of cell surface receptors and may affect a variety of intracellular signalling systems including phosphollipase C, phospholipase A₂, potassium channels and calcium channels (Skene, J.H.P., et al .. Science 233:783 (1986); Stryer, L., et al.. Ann. Rev. Cell Biol. 2:391 (1986); Neer, E.J., Nature 333:129 (1988); Ross, E.M., Neuron 3:141 (1989); Brown, A.M., et al.. Am. J. Phvsiol. 254:H401 (1988)). The strikingly high levels of G₀ 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 G₀-based transduction system in growth cones.

G proteins have been proved crucial to developmental morphogenesis in the slime mold, Dictvostelium. where chemotaxis towards cAMP is transduced via a G protein (Snaar-Jagalska, B.E., et al.. F.E.B.S. Lett. 232:148 (1988); Snaar- Jagalska, B.E., et al., F.E.B.S. Lett. 236:139 (1988); Kesbeke, F., et al.. J. Cell Biol. 107:521 (1988)). Similarly, signals from pathways or targets in the developing nervous system may bind to a G₀-linked receptor, or receptors, in the growth cone membrane, and thereby alter the level of intracellular second messengers, and hence growth cone motility. In general, these signals, receptors, and second messengers are unknown at present. One class of candidate receptors are the cell adhesion molecules, N-CAM and L1, which are localized to the neuronal growth cone (Letourneau, P.C, 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, C.O.M., et al.. J. Cell Biol. 108:1115 (1989)).

The persistence of G₀ expression in the adult nervous system (Worley, P.F., et al .. Proc. Natl. Acad. Sci. USA 83:4561 (1986)) implies roles other than the regulation of axonogenesis. Another growth cone enriched molecule, GAP-43, also exists in discrete regions of the adult brain (Benowitz, L.I., et al.. Trends Neurosci. 10:527 (1987); Skene, H.J.P., Ann. Rev. Neurosci. 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, A., N.Y. Acad. Sci. 444:980 (1989)) has suggested a role for GAP-43 in synaptic plasticity in the adult (Benowitz, L.I., et al., Trends Neurosci . 10:527 (1987); Skene, H.J.P., Ann. Rev. Neurosci . 12:127 (1989)). It is noteworthy that pertussis toxin blocks long-term potentiation, perhaps implicating G₀ in this process as well (Goh, J.W., Science 244:980 (1989)). Hence, G₀ may transduce regulatory signals for axonal extension during neuronal development and for synaptic plasticity in the adult nervous system. EXAMPLE IX

GAP-43 is a Novel Internal Regulator of Protein Binding

In another aspect, the present invention is directed to the surprising discovery that GAP-43 acts within the cell to modify the binding capacity of other cell proteins, including that of G₀. As far as the present inventors are aware, this constitutes the first report of an important new class of internal regulatory proteins ("IRP"), of which GAP-43 is representative, comparable in effect and utility to external cell receptors. Those of skill will easily recognize that the IRP compositions and methods of this aspect of the invention allow the internal modulation of protein activity, and, thereby, of cell activity and function, in neuronal and non-neuronal cells.

Further, it has surprisingly, been found that synthetic peptides comprising the amino terminus amino acids of GAP-43 duplicate exactly the modulation in GTP binding by G₀ that is caused by the intact GAP-43 protein. Synthetic IRP peptides according to this embodiment comprise the following sequences:

I. MLCCMRRTKQVEKNDEDQKIEQDGV;

II. MLCCMRRTKQVEKNDEDQKIEQDG;

III. MLCCMRRTKQVEKNDEDQKIEQD;

IV. MLCCMRRTKQVEKNDEDQKIEQ;

V. MLCCMRRTKQVEKNDEDQKIE;

VI. MLCCMRRTKQVEKNDEDQKI;

VII. MLCCMRRTKQVEKNDEDQK;

VIII. MLCCMRRTQVEKNDEDQ;

IX. MLCCMRRTKQVEKNDED;

X. MLCCMRRTKQVEKNDE;

XI. MLCCMRRTKQVEKND; XII. MLCCMRRTKQVEKN;

XIII. MLCCMRRTKQVEK;

XIV. MLCCMRRTQVE;

XV. MLCCMRRTKQV;

XVI. MLCCMRRTKQ;

XVII. MLCCMRRTK;

XVIII. MLCCMRRT;

XIX. MLCCMRR;

XX. MLCCMR;

XXI. MLCCM;

XXII. MLCC; and

XXIII. functional derivatives thereof.

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 hydrophobic-leu-cys-cys-x-basic-basic or functional derivatives thereof. It will be further appreciated from the present teachings that the cysteines of the IRPs and IRP peptides of the invention may be prone to palmitylation.

Those of skill also will appreciate that, by varying the structure of the IRP proteins and peptides of the invention, the target protein activity may be enhanced or, if desired, inhibited in an unprecedented manner. Thus, in another embodiment, there is provided 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, G₀. The preferred binding substrate is GTP, and GTPγS is most preferred. The environment is preferably that inside a living cell, which may be a central or peripheral neural cell.

Those of skill will appreciate that the methods of the invention may be carried out in vitro, in situ, or in vivo, with the latter being most preferred, keeping in mind the generally accepted principles of administration well known in the art, as discussed herein.

Those of skill who have the benefit of the teachings of the invention will appreciate that internal regulation of protein activity offers significant opportunities for the efficacious treatment of disorders in mammals, including humans, and that such treatment is especially valuable in preventing, ameliorating, or reversing the effects of neural disease or dysfunction, inasmuch as the 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. Also, by such means, it is possible to regulate the activity of a desired protein with an exquisite degree of control. Thus, in another aspect, 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. In another aspect, 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. Use of the 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.

In another aspect, 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.

In yet another embodiment of the invention, the GAP-43 sequences of the invention have been used to isolate a G-like protein from neural cells. Using a GAP-43 column, 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 CaCl₂, and 1 mM MgCl₂. The protein elutes in a single band with 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. It also will be appreciated that 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 G₀ is enhanced when considered in conjunction with the demonstration herein that G₀ is a major noncytoskeletal protein present in neuronal growth cones. The discovery that GAP-43, the function of which has not previously been known, modulates G₀ activity, is evidence that GAP-43 is a long sought "missing link" between first and second messengers in cellular transduction systems. Although not wishing to be bound by any particular theory, it may be that persistent activation of G₀ by GAP-43 could make the cells ignore their environment and hence grow constitutively. The present work also suggests that there is a family of other molecules that contain sequences similar to the amino terminus of GAP-43, and hence regulate G₀ from inside the cell. It is of great interest that the amino terminus of GAP-43 bears a significant resemblance to the cytosolic domains of several G protein-linked receptors (such as the beta receptor). This suggests that GAP-43 may interact with G₀ in a similar place in the molecule as do the cytosolic domains of the G protein-linked receptors. METHODS AND RESULTS

GAP-43 Stimulates GTPΎS Binding to G₀.

With the major growth cone membrane proteins identified, the inventors sought to determine whether these components were capable of forming intermolecular complexes. In particular, G₀ and GAP-43 were examined since they are the major non-cytoskeletal proteins in the growth cone membrane. Attempts to physically isolate a GAP-43/G₀ complex by gel exclusion chromatography, immunoprecipitation and affinity chromatography were unsuccessful.

To identify transient GAP-43/G₀ interactions in solution, GTPγS (guanidine triphosphate gamma S³⁵) binding to purified G₀ was measured in the presence of varying concentrations of purified GAP-43. GAP-43 itself does not binding GTPγS, but GAP-43 stimulates GTPγS binding to G₀ by 160 + 30%. This effect is saturable and is half maximal in the presence of 150 nM GAP-43. The concentration of both G₀ and GAP-43 in whole brain is on the order of 2 μm, so that this affinity 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 GTPγS binding. G₀ is known to be partially inactivated during preincubation at 30ºC without GTPγS. GAP-43 was found not to affect the degree of G₀ thermal instability.

Ligand-receptor complexes which interact with G proteins stimulate GTPγS binding to approximately the same extent as GAP-43. Their effect is blocked by pertussis toxin treatment of G₀. Similarly, pertussis toxin treatment of G₀ abolishes the GAP-43 action.

GAP-43 has previously been shown to bind calmodul in, an interaction which is enhanced in the absence of calcium. The physiologic relevance of this association is unclear. The addition of calmodul in to G₀ causes no change in the ability of GAP-43 to stimulate GTPγS binding, with or without calcium.

A GAP-43 Decapeptide Interacts With G₀.

To determine which regions of GAP-43 were critical for stimulation of GTPγS binding to G₀, a series of synthetic peptides from the GAP-43 sequence were tested. Peptides containing either the first 24 amino acids or the first 10 amino acids of GAP-43 stimulate GTPγS binding with an EC50 of 20μM. In contrast, peptides from three other regions of GAP-43 are ineffective at higher concentration. The amino terminal peptides stimulate GTPγS binding to the same level as does GAP-43 itself, and the addition of GATP-43 protein together with amino terminal peptide does not stimulate binding to higher levels. This suggests that the peptide is a full competitive agonist for GAP-43.

In a previous example, it was shown that this same region of GAP-43 is necessary and sufficient for its membrane association, and that the two cysteines are critical. To determine whether the same cysteine dependence exists for G₀ stimulation, a decapeptide with threonines substituted for the two cysteines was synthesized. This peptide fails to stimulate GTPγS binding to G₀.

The other group of proteins known to stimulate GTPγS binding to G proteins are hormone and neurotransmitter receptors. Although their overall structure, with a large extracellular region and seven transmembrane domains, is much different from that of GAP-43, the inventions nevertheless searched for homologies between these proteins and the amino terminus of GAP-43. The interaction of the β₂-adrenergic receptor with G_s is interrupted most specifically by a point mutation at a palmitylated cysteine in the cytoplasmic tail of the protein. The cysteines in the amino terminus of GAP-43 are also palmitylated. There is a consensus sequence shared by GAP-43 and these receptors which consists of hydrophobicleu-cys-cys-x-basic-basic, where the cysteines are prone to palmityl ation. DISCUSSION

The present data provide a growth cone mechanism for the coordination of extracellular signals with the expression of intracellular growth associated proteins during neuronal morphogenesis. The strikingly high levels of the alpha and beta subunits of G₀ in the growth cone membrane suggest a major role for G₀ in neurite regulation. The G₀ concentration in the growth cone membrane exceeds that of another G protein, transducin, in the highly specialized outer segment of retinal photoreceptor cells.

In systems where G protein function is clearly defined, 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. There are many heterotrimeric alpha-beta-gamma G proteins, differing primarily in their alpha subunits. In general, 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. G₀ is predominantly expressed in brain, where it is the major form of G protein. In adults, it is found in the neuropil (-), and our data localize G₀ to the tips of neurites in growing cells where it is the major non-cytoskeletal protein . G₀ 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 A₂. There is evidence that some matrix and soluble effects on the growth cone involve G-protein transduction. Antibodies to the growth cone localized cell adhesion proteins, N-CAM and LI, alter calcium levels and phosphotidylinositol metabolism in PC12 cells. The effect of these antibodies is blocked by the G₀/G_i inhibitor pertussis toxin (Schuch, U., et al., Neuron 3:13 (1989)). In certain heliosome neurons, serotonin, which acts via a G-coupled receptor, is a potent inhibitor of neurite extension.

The restricted localization of G₀ 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 wondered whether GAP-43 might interact with G₀. GAP-43 enhances GTPγS binding to G₀. Furthermore, a small region of GAP-43, defined by a synthetic decapeptide, exerts this action. Stimulation of GTPγS binding by GAP-43 is similar to that by agonist-receptor complexes, and the decapeptide sequence has homology with these receptors. Although not intending to be bound by any particular theory, the most likely interpretation is that, in vivo. GAP-43 mimics transmembrane receptors and activates G₀, creating a GTP-bound alpha subunit which then triggers an intracelluiar second messenger system. Alternatively, GAP-43 binding to G₀ might function primarily to disrupt G₀-receptor or G₀-effector interactions. It is also conceivable that GAP-43 is an effector of G₀ activation by receptor in some as yet unknown manner.

The modulation of a G₀ cone transduction system by a growth associated protein, GAP-43, provides a mechanism to integrate extracellular signals with an intracellular program for neuronal growth. Further regulation of the system could occur via other modifications, such as phosphorylation of the receptor by receptor kinases such as BARK, or phosphorylation of GAP-43 by protein kinase C. In this model, GAP-43 might synergistically enhance the response of G₀ to extracellular ligands, or decrease responsiveness to ligands by overriding the dependency of G₀ 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 GTPγS binding to G₀ are subject to palmitylation. In our experiments, the amino terminal peptides and probably the GAP-43 (prepared by pH 11 extraction of membranes) 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 palmitylation-depalmitylation plays a regulatory role for these proteins.

The persistence of G₀ expression in the adult nervous system (Worley, P.F., et al., Proc. Natl. Acad. Sci. USA 83:4561 (1986)) implies roles other than the regulation of neurite outgrowth during development and regeneration. GAP-43 also exists in discrete regions of the adult brain, and the immunohistochemical maps for the two proteins are strikingly similar, if the cerebellum is excluded (Benowitz, I . I . et al.. Trends Neurosci. 10:527 (1987); Skene, H.J.P., Ann. Rev. Neurosci. 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, A., Ann. N.Y. Acad. Sci. 444:203 (1985)) has suggested a role for GAP-43 in synaptic plasticity in the adult (Benowitz, I.I., et al., Trends Neurosci. 10:527 (1987); Skene, H.J.P., Ann. Rev. Neurosci. 12:127 (1989)). It is noteworthy that the G₀/G_i antagonist, pertussis toxin, blocks long-term potentiation, perhaps implicating G₀ in this process as well (Goh, J.W., et al., Science 244:980 (1989)). Hence, G₀ may transduce intracellular and extracellular signals for neurite extension during development and for synaptic plasticity in the adult nervous system. DEPOSIT 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). As an additional embodiment, 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 .

It will be appreciated by those of skill upon reviewing the preceeding description of the preferred embodiments, including the examples presented herein, that 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. It will further be appreciated that 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 structural genes, using methods well known to those of skill.

Thus, stated more broadly, 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. Structural genes, or fragments thereof, comprising, at their amino-terminus end, in phase, the nucleotide sequence of the GAP-43 promoter as described herein, and functional or chemical derivatives thereof, also form intended embodiments of the invention.

In yet additional embodiment, there are provided DNA expression vectors comprising the structural gene as described above, host cells transfected with these vectors, and the proteins produced thereby.

Claims

CLAIMS What is claimed is:

1. Substantially pure mammalian GAP-43, or a functional derivative thereof.

2. The composition of claim 1, wherein said mammalian GAP-43 is rat GAP-43.

3. The composition of claim 1, wherein said mammalian GAP-43 is human GAP-43.

4. The composition of claim 2, wherein said rat GAP-43 has an amino acid sequence as shown in or substantially similar to that shown in Figure 2.

5. The composition of claim 3, wherein said human GAP-43 has an amino acid sequence as shown in or substantially similar to that shown in Figure 5A.

6. A polypeptide comprising an amino acid sequence as shown in or substantially similar to that shown in Figure 2, or a functional derivative thereof.

7. A polypeptide comprising an amino acid sequence as shown in or substantially similar to that shown in Figure 5A, or a functional derivative thereof.

8. cDNA comprising a nucleotide sequence as shown in or substantially similar to that shown in Figure 2, or a functional derivative thereof.

9. cDNA comprising a nucleotide sequence as shown in or substantially similar to that shown in Figure 5A, or a functional derivative thereof.

10. A DNA expression vector comprising the cDNA of claim 8.

11. A DNA expression vector comprising the cDNA of claim 9.

12. A host cell transfected with the vector of any of claims 10 or 11.

13. The host cell of claim 12, wherein said cell is selected from the group consisting of prokaryotic cells and eukaryotic cells.

14. GAP-43 produced by the cell of claim 13, or a functional derivative thereof.

15. A method of producing mammalian GAP-43 or a functional derivative thereof, comprising transfecting a prokaryotic or eukaryotic host cell with a vector comprising cDNA encoding mammalian GAP-43, culturing said host cell in a suitable medium under conditions permitting expression of said mammalian GAP-43, and separating said mammalian GAP-43 from said medium.

16. Antibody directed against mammalian GAP-43, or a functional or chemical derivative thereof.

17. Antibody directed against rat GAP-43, or a functional or chemical derivative thereof.

18. Antibody directed against human GAP-43, or a functional or chemical derivative thereof.

19. The antibody of any of claims 16, 17 or 18, wherein said antibody is selected from the group consisting of monoclonal antibody and polyclonal antibody.

20. The antibody of claim 19, wherein said antibody is detectably labeled.

21. The antibody of claim 19, wherein said antibody is therapeutically labeled.

22. A pharmaceutical composition comprising the composition of any of claims 1,2,3,4,5,6,7 or 14, together with a pharmaceutically acceptable carrier.

23. The composition of claim 22, additionally comprising one or more therapeutically effective agents.

24. A method of determining or detecting mammalian GAP-43 in a sample, comprising contacting a sample suspected of containing GAP-43 with the antibody of claim 20, incubating said sample with said antibody so as to allow the formation of a GAP-43 antibody complex, separating complexed antibody from uncomplexed antibody, and detecting the labeled complexed antibody.

25. A kit useful for the determination or detection of GAP-43, 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 detectably labeled antibody to GAP-43.

26. The kit of claim 25, wherein said antibody is selected from the group consisting of polyclonal and monoclonal.

27. A method of inducing expression of GAP-43 in cells, comprising exposing said cells to an effective amount of nerve growth factor.

28. The method of claim 27, wherein said cells are neural cells.

29. The method of claim 28, wherein said cells are exposed to said nerve growth factor in situ.

30. A method of enhancing expression of GAP-43 in cells, comprising introducing into said cells a DNA expression vector comprising cDNA encoding GAP-43.

31. The method of claim 30, wherein said vector is introduced into said cells by transfection, transduction, or direct microinjection.

32. A method of promoting structural remodeling in neural cells, comprising inducing GAP-43 expression in said cells by the method of any of claims 28, 29, 30 or 31.

33. A method of promoting healing of damaged neural tissue, comprising inducing GAP-43 expression in and around said damaged neural tissue.

34. The method of claim 33, wherein said neural damage is caused by infarction with ischemia, transient ischemia without infarction, hypoxia, anoxia, anoxic encephalopathy, hypoperfusion, or stroke.

35. A method of modulating structural remodeling in 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.

36. A method of modulating synaptic plasticity in 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.

37. A method of modulating the microenvironment of 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.

38. A method of inhibiting GAP-43 expression in mammalian neuronal cells, comprising exposing said cells to an effective amount of one or more steroids.

39. A method of modulating GAP-43 expression in fully differentiated mammalian neuronal cells, comprising exposing said cells to an effective amount of one or more steroids.

40. The method of any of claims 35, 36, 37, 38 or 39, wherein said steroid is a corticosteroid.

41. The method of claim 41, wherein said corticosteroid is selected from the group consisting of mineralocorticoid and glucocorticoid.

42. The method of claim 41, wherein said mineralocorticoid is selected from the group consisting of dexamethasone, corticosterone, aldosterone and progesterone.

43. A method of augmenting steroidal inhibition of GAP-43 expression in mammalian neuronal cells exposed to steroids, comprising exposing said cells to an effective amount of cycloheximide.

44. The method of any of claims 30 or 31, wherein said cells are non-neuronal cells.

45. cDNA encoding a membrane-targeting peptide comprising the nucleotide sequence

atg ctg tgc tgt atg aga aga ace aaa cag or a functional derivative thereof.

46. A membrane-targeting peptide comprising an amino acid sequence selected from the group consisting of

I. MET LEU CYS CYS MET ARG ARG THR LYS GLN;

II. MET LEU CYS CYS MET ARG ARG THR LYS;

III. MET LEU CYS CYS MET ARG ARG THR;

IV. MET LEU CYS CYS MET ARG ARG;

V. MET LEU CYS CYS MET ARG;

VI. MET LEU CYS CYS MET;

VII. MET LEU CYS CYS; and

VIII. functional derivatives thereof.

47. A DNA sequence encoding a membrane-targeting peptide comprising nucleotides encoding an amino acid sequence selected from the group consisting of

I. MET LEU CYS CYS MET ARG ARG THR LYS GLN;

II. MET LEU CYS CYS MET ARG ARG THR LYS;

III. MET LEU CYS CYS MET ARG ARG THR; IV. MET LEU CYS CYS MET ARG ARG;

V. MET LEU CYS CYS MET ARG;

VI. MET LEU CYS CYS MET;

VII. MET LEU CYS CYS; and

VIII. functional derivatives thereof.

48. A structural gene or fragment thereof, comprising, at its amino-terminus end, in phase, nucleotides encoding a membrane-targeting peptide having the sequence of claim 46.

49. A protein or peptide comprising, at its aminoterminus end, a membrane-targeting peptide comprising the sequence of claim 46.

50. A method for directing a desired protein or peptide to the membrane of a cell, comprising

(a) ligating to the amino-terminus of said protein or peptide a membrane-targeting peptide comprising the amino acid sequence of claim 46; and

(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 membranetargeting domain.

51. The method of claim 50, wherein said cell is selected from the group consisting of neuronal and non-neuronal cells.

52. The method of claim 51, wherein in said neuronal cell said resulting protein or peptide of step (b) is directed to the growth cone region of said cell.

53. A monoclonal antibody having substantially the specificity of MAb anti-GAP-43 (H5) or a functional or chemical derivative thereof, said MAb anti-GAP-43 (H5) produced by hybridoma strain H5, said hybridoma strain having accession number ATCC _ .

54. Hybridoma strain H5, having accession number ATCC _, or a functional or chemical derivative thereof.

55. A nucleotide sequence as shown in Figure 13 encoding genomic GAP-43, or a functional or chemical derivative thereof.

56. A nucleotide sequence as shown in Figure 14 encoding the GAP-43 promoter, or a functional or chemical derivative thereof.

57. 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.

58. A structural gene or fragment thereof, comprising, at its amino terminus end, in phase, the nucleotide sequence of claims 56 or 57, or a functional or chemical derivative thereof.

59. A DNA expression vector comprising the structural gene of claim 58.

60. A host cell transfected with the vector of claim 59.

61. An Internal Regulatory Protein (IRP).

62. An IRP peptide comprising an amino acid sequence selected from the group consisting of:

I. MLCCMRRTKQVEKNDEDQKIEQDGV;

II. MLCCMRRTKQVEKNDEDQKIEQDG;

III. MLCCMRRTKQVEKNDEDQKIEQD;

IV. MLCCMRRTKQVEKNDEDQKIEQ;

V. MLCCMRRTKQVEKNDEDQKIE;

VI. MLCCMRRTKQVEKNDEDQKI;

VII. MLCCMRRTKQVEKNDEDQK;

VIII. MLCCMRRTQVEKNDEDQ;

IX. MLCCMRRTKQVEKNDED;

X. MLCCMRRTKQVEKNDE;

XI. MLCCMRRTKQVEKND;

XII. MLCCMRRTKQVEKN;

XIII. MLCCMRRTKQVEK;

XIV. MLCCMRRTQVE;

XV. MLCCMRRTKQV;

XVI. MLCCMRRTKQ;

XVII. MLCCMRRTK;

XVIII. MLCCMRRT;

XIX. MLCCMRR;

XX. MLCCMR;

XXI. MLCCM;

XXII. MLCC; and

XXIII. functional derivatives thereof.

63. A nucleotide sequence encoding the IRP peptide of claim 62.

64. An IRP peptide having the consensus amino acid sequence

hydrophobic-leu-cys-cys-x-basic-basic

or a functional derivative thereof.

65. The IRP peptide of claims 62 or 64, wherein the cysteines are prone to palmitylation.

66. 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.

67. The method of claim 66, wherein the desired protein is a G protein.

68. The method of claim 67, wherein said G protein is G₀.

69. The method of claim 66, wherein said IRP peptide is the peptide of claims 62, 64 or 65.

70. The method of claim 66, wherein said environment is inside a living cell.

71. The method of claim 70, wherein said cell is a central or peripheral neural cell.

72. The method of claim 67, wherein said binding substrate is GTP.

73. A monoclonal antibody directed against the IRP peptide of claim 62 or 64, or a functional or chemical derivative thereof, said monoclonal antibody or its said derivative optionally detectably or therapeutically labeled.

74. A pharmaceutical composition comprising the IRP peptide of claim 62 or 64, together with a pharmaceutically acceptable carrier, and optionally comprising one or more therapeutically effective agents.

75. A pharmaceutical composition comprising the monoclonal antibody of claim 73, together with a pharmaceutically acceptable carrier, and optionally comprising one or more therapeutically effective agents.

76. A method of modulating structural remodeling in a neural cell, comprising administering to said cell an effective amount of the composition of claim 74 or 75.

77. A neural growth-associated protein, characterized in that it binds specifically to GAP-43, has a molecular weight of 39,000, elutes in a single band with application of EDTA in 50 mM Tris buffer, and is non-reactive with polyclonal antibody to G₀.