MXPA99001282A - Neuritin, a neurogene - Google Patents

Abstract

Disclosed are DNA and amino acid sequences for a novel polypeptide termed Neuritin which is expressed primarily in selected regions of the brain.

Description

NEURITINE, A NEUROGEN BACKGROUND Field of the Invention This invention relates to the novel DNA sequences that encode a polypeptide called Neuritin, which is expressed mainly in certain brain tissues in response to certain stimuli.

Related Technique A number of neurological disorders and diseases are caused at least in part by the degeneration or death of particular classes of neurons. For example, Parkinson's disease is characterized by delayed voluntary muscle movement, muscle stiffness, and tremor. Such symptoms are attributed at least in part to the progressive degeneration of dopamine-producing neurons located in a specific region of the brain called the substantia nigra. The degeneration of these neurons REF .: 29461 ("dopaminergic neurons") results in a decrease in dopamine levels in an adjacent region of the brain called the striatum. The striatum contains neurons that express receptors for dopamine / these neurons are involved in the control of motor activity. The cause of the degeneration of dopaminergic neurons is unknown, but it has been attributed to free radicals, excess iron content, environmental toxins, excitatory amino acid neurotoxicity, and possibly a deficiency of certain neurotrophic factors (Jenner, Ne urol ogy, Suppl 3: Sß-S12 [1995], Adams and Victor, eds Prin cipy of Ne urolgy, Chapter 42: Degenerative Diseases of the Nervous System, McGraw Hill, NY [1993]). Diseases such as amyotrophic lateral sclerosis (ALS), progressive muscular atrophy, and hereditary motor and sensory neuropathy (Charcot-Marie-Tooth disease) all result at least in part from a decline in motor neurons which are localized in the ventral horn of the spinal cord.

The hippocampus, a well-defined structure that is part of the cerebral cortex of the brain, is important in the formation of long-term memory. The destruction of the hippocampus, for example by ischemia, can result in an inability to form new memories. The degeneration of CAÍ pyramidal neurons, which are located in the CAI region of the hippocampus, is a characteristic of Alzheimer's disease. These same neurons are selectively vulnerable to ischemic and anoxic damage that occurs in conditions such as stroke and brain trauma. In addition, CAI pyramidal hippocampal neurons, as well as pyramidal neurons located in the CA3 region of the hippocampus, are selectively damaged in epilepsy. The striatum is the region of innervation of the nerve terminals of neurons containing dopamine from the substantia nigra. Most striatal neurons use GABA (4-aminobutyric acid) as their neurotransmitter. The striated body is. the main objective of the progressive neurodegeneration that occurs in Huntington's disease, in which the greatest loss of neurons is that of the neurons that use the GABA of the striatum. The neurons that contain serotonin are located in groups accumulated around the midline of the metencephalon. These neurons are involved in the control of body temperature, mood and sleep. Disorders of the neuronal system containing serotonin include, for example, depression, other mood disorders and sleep disorders. Photoreceptor cells are a specialized subset of retinal neurons, and are responsible for vision. Damage and / or death of photoreceptor cells can lead to blindness. Retinal degeneration, such as retinitis pigmentosa, age-related macular degeneration, and stationary night blindness, are all characterized by progressive atrophy and loss of function of the photoreceptor exterior segments, which are specialized structures containing the visual pigments that transform a light stimulus into electrical activity.

While there are some therapies available to treat the symptoms and decrease the severity of such diseases (for example, L-dopa to treat Parkinson's disease), there is currently no effective treatment to prevent or reduce the degeneration of most classes. previously mentioned neurons affected, or to promote their repair. Recently, several protein molecules of natural origin have been identified based on their trophic activity on various neurons. These molecules are called "neurotrophic factors". The neurotrous factors are soluble, endogenous proteins that can regulate the survival, growth and / or morphological plasticity of neurons (see Fallón and Laughlin, Neuro trophi c Fa ct ors, Academic Press, San Diego, CA [1993]. ]). The known neurotrophic factors belong to various different proteins superfamilies of the idle polypept growth factors, based on their amino acid sequence homology and / or their three-dimensional structure (MacDonald and Hendrikson, Cell 1, 73: 421-424 [ 1993]). A family of neurotrophic factors is the neurotrophin family. This family currently consists of NGF (nerve growth factor), BDNF (neurotrophic factor derived from the brain), NT-3 (neurotrophin 3), NT-4 (neurotrophin 4), and NT-6 (neurotrophin 6). CNTF (ciliary neurotrophic factor) and LIF (Leukemia inhibitory factor) are cytokine polypeptides that have neurotrophic activity. By virtue of their structural characteristics and receptor components, these polypeptides are related to a family of hematopoietic cytokines that includes IL-6 (int erleucine 6), IL-11 (interleukin 11), G-CSF (factor of stimulation of granulocyte colonies), and oncostatin M. GDNF (glial-derived neurotrophic factor) is a neurotrophic factor that belongs to the TGF-beta superfamily (transforming growth factor beta). GDNF shows potent promoter actions of survival and differentiation for dopaminergic and motor neurons (Lin et al., Sci en ce, 260: 1130-1132 [1993]; Yan et al., Na ture, 373: 341-344 [1995]. ]).

While it is known that these neurotrophic factors increase the development and / or survival of neurons, less is known about the molecules that work in conjunction with these factors. One way in which additional neurotrophins and related molecules can be identified is to administer to an animal one or more compounds that are known to have an effect on the nervous system, and then analyze the tissues for the induction of genes involved in the responses neuronal to the compounds. For example, genes that are induced in certain tissues of the nervous system, such as the hippocampal region of the brain, can be selected. This technique was used by Nedivi and collaborators (Na ture, 363: 718-722 [1993]; Nedivi et al, Proc. Na ti, Acad.Sci USA, 93: 2048-2053 [1996]) to identify new genes that are induced from the dentate gyrus. of the hippocampus, in response to the administration of a glutamate neurotransmitter analog, called kainate (kainic acid). The expression of many neurotrophic factors such as NGF, BDNF, NT3, GDNF, bFGF, IGF-1 and TGF-beta is regulated by afferent neuronal activity and / or by neuronal damage. The strong induction of some of these genes can be observed in the dentate gyrus of the hippocampus, in response to the glutamate analog, kainate (Isackson, Curren t Opinions on Neurobiology 5: 50-357 [1995]). Treatment with kainate appears to increase the release of novel compounds from the hippocampus of alert rats, and this activity appears to be different from the actions of known neurotrophic factors (Humpel et al., Sci en ce, 269: 552-554 [1995]. ]). In view of the fact that many disorders and diseases of the nervous system have no known cure, there is a need in the art to identify new compounds for the treatment of neurological conditions and neurological diseases such as Parkinson's disease, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, seizures, and various degenerative disorders that affect vision Accordingly, an objective of the present invention is to provide the novel compounds that may be useful in the promotion of neuronal regeneration and the restoration of neural functions. A further objective of the present invention is to provide a method for the treatment of certain neurological diseases. These and other objects will be apparent to one of ordinary skill in the art from the present description.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides a nucleic acid molecule encoding a polypeptide selected from the group consisting of: a) the nucleic acid molecule of SEQ ID NO. 1; b) the nucleic acid molecule of SEQ ID NO. 2; c) a nucleic acid molecule encoding the polypeptide of SEQ ID NO. 3; d) a nucleic acid molecule encoding the polypeptide of SEQ ID NO. 4; e) a nucleic acid molecule encoding a polypeptide that is at least 70 percent identical to the polypeptide of SEQ ID NO. 3 or of SEQ ID NO. 4; and f) a nucleic acid molecule that is the complement of any of (a) - (e) above. In yet another embodiment, the present invention provides the vectors comprising the nucleic acid molecules described above. In still another embodiment, the present invention provides the host cells comprising these vectors. In a further embodiment, the present invention provides a process for the production of a neuritin polypeptide comprising the steps of: a) expressing a polypeptide encoded by the nucleic acid of claim 1 in a suitable host; and b) isolation of the polypeptide. Optionally, the Neuritin polypeptide is SEQ ID NO. 3 or SEQ ID NO. 4. In yet another embodiment, the present invention provides a Neuritin polypeptide selected from the group consisting of: a) the polypeptide of SEQ ID NO. 3; b) the polypeptide of SEQ ID NO. 4; and c) a polypeptide that is at least 70 percent homologous with the polypeptide of (a) or (b). Optionally, the Neuritin polypeptide can be a biologically active fragment of Neuritin, such as amino acids 25-115, 25-143, amino acids 1-115, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 describes a rat Neuritin cDNA sequence (SEQ ID NO: 1).

Figure 2 depicts a human Neuritin cDNA sequence (SEQ ID NO 2).

Figure 3 depicts the full-length, translated amino acid sequence for rat Neuritin (SEQ ID No. 3).

Figure 4 describes the full length, translated, amino acid sequence for human Neuritin (SEQ ID NO: 4). Figure 5 depicts two Northern blots. Figure 5A is a Northern blot of various rat tissues probed with a Neuritin probe. The abbreviations are h (heart); br (brain); sp (spleen); lu (lung); li (liver); m (muscle); k (kidney); t (testicles). Figure 5B is a Northern blot of various brain regions of either control rats (-) or rats treated with kainic acid (+). The abbreviations are Cereb (cerebellum); Hipp '(hippocampus); DG (dentate convolution). The spot was probed with a Neuritin probe. Figure 6 describes various Northern blotches. Figure 6A shows Northern blotting of hippocampal and rat cortical neurons treated with BDNF, NT-3, FGF, AMPA, NMDA, or KCl. The "0" controls did not receive treatment. Figure 6B shows a Northern blot of hippocampal RNA and bark obtained from rats injected with saline ("S") or BDNF ("B"). "0" indicates without treatment. Figure 7 is a graph of the induction over time of Neuritin mRNA levels of rat hippocampal neurons E-18, in response to treatment with either BDNF or KCl.

Figure 8 describes two Western blots probed with an antibody against Neuritin. Figure 8A depicts a Western blot of CHO cells transfected with either the control plasmid ("progenitor") or the plasmid containing the gene encoding full-length human Neuritin (cell line designated "CHO 15.4"). "PI-PLC" refers to phosphinositol-phospholipase C, and "+" and "-" refers to the presence or absence of PI-PLC.Figure 8B describes a Western blot of various rat tissues. was probed with an antibody against Neuritin, abbreviations for the tissues evaluated in this spotting are found in the text, Figure 9 describes cultures of rat embryonic neurons, hippocampals.

("Hipp") and cortical ("Cort") incubated in the presence (+) or absence (-) of Neuritin. "Dil" refers to the treatment with the lipophilic fluorescent dye Dil.

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "Neuritin" when used to describe a nucleic acid molecule refers to a nucleic acid molecule or fragment thereof that (a) has the nucleotide sequence as described in SEQ ID. DO NOT. 1 or SEQ ID NO. 2; (b) has a nucleic acid sequence that codes for a polypeptide that is at least 70 percent identical, but can be at least 80 percent or 90 percent identical, to the polypeptide encoded by any of SEQ ID NOS. 1 or 2; (c) is an allelic variant of natural origin of (a) or (b); (d) is a variant nucleic acid of (a) - (c) produced as provided herein; and / or (e) is complementary to (a) - (d). Percent sequential identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal coupling of their respective amino acids (either along the entire length of one or both sequences, or along a predetermined portion of one or both sequences). The programs provide a "default" opening penalty and an "empty" space penalty, and a qualification matrix such as PAM 250 (a standard rating matrix; see Dayhoff et al., In Atlas of Protein Sequence and Structure). , vol.5, supp.3 [1978]) can be used in conjunction with the computer program. The percentage identity can then be calculated as: Total number of identical links X 100 [length of the longest sequence within the coupled section] + [number of empty spaces entered within the longest sequence, in order to align the two sequences] Polypeptides that are at least 70 percent identical will typically have one or more substitutions, deletions and / or amino acid insertions. . Usually, substitutions will be conservative to have little or no effect on the complete net charge, polarity, or hydrophobicity of the protein, but may optionally increase the activity of Neuritin. Conservative substitutions are described in Table I below.

Table I Conserved Basic amino acid substitutions Arginine Lysine Histidine Acids Glutamic acid Aspartic acid Polar Glutamine Asparagine Hydrophobic Leucine Isoleucine Valine Aromatics Phenylalanine Tryptophan Tyrosine Small: Glycine Alanine Serine Threonine Methionine The term "stringent conditions" refers to hybridization and washing under conditions that they only allow the binding of a nucleic acid molecule such as an oligonucleotide or probe of cDNA molecule to highly homologous sequences. A stringent wash solution is 0.015 M sodium chloride, 0.005 M sodium citrate, and 0.1 percent SDS used at a temperature of 55 ° C-65 ° C. Another strict wash solution is 0.2 x SSC and 0.1 percent SDS used at a temperature between 50 ° C and 65 ° C. Where olonuclet probes are used to select genomic or cDNA libraries, the following strict wash conditions can be used. One protocol uses 6 x SSC with 0.05 percent sodium pyrophosphate at a temperature of 35 ° C-62 ° C, depending on the length of the oligonucleotide probe. For example, probes of 14 base pairs are washed at 35-40 ° C, probes of 17 base pairs are washed at 45-50 ° C, probes of 20 base pairs are washed at 52-57 ° C, and probes of 23 base pairs are washed at 57-63 ° C. The temperature can be increased from 2 to 3 ° C where the previous non-specific binding appears high. A second protocol uses tetramethylammonium chloride (TMAC) to wash oligonucleotide probes. A strict washing solution is TMAC 3M, 50 M Tris-HCl, pH 8.0, and 0.2 percent SDS. The wash temperature using this solution is a function of the length of the probe. For example, a probe of 17 base pairs is washed at approximately 45-50 ° C. The term "Neuritin protein" or "Neuritin polypeptide" as used herein refers to any protein or polypeptide having the properties described herein for Neuritin. The Neuritin polypeptide may or may not have an amino-terminal methionine, depending on the manner in which it is prepared. By way of illustration, the Neuritin protein or the Neuritin polypeptide refers to 1) an amino acid sequence encoded by the nucleic acid molecule described in any of the above (a) - (e), and the peptide or fragments polypeptides derived therefrom, 2) the amino acid sequence described in SEQ ID NOS: 3 or 4, and / or 3) the chemically modified derivatives as well as the variants of the nucleic acid and / or amino acid sequence of the themselves, as provided herein. As used herein, the term "Neuritin fragment" refers to a peptide or a polypeptide that is less than the full length amino acid sequence of the naturally occurring Neuritin protein, but has substantially the same biological activity as the protein. Neuritin polypeptide or the Neuritin protein described above. Such a fragment can be truncated at the amino terminus, the carboxyl terminus (such as the GPI anchor domain which is approximately the last 27 amino acids of the Neuritin polypeptide), and / or internally, and can be chemically modified. Preferably, the Neuritin fragment will be one that retains at least all 6 cysteine residues. Such Neuritin fragments can be prepared with or without an amino-terminal methionine. As used herein, the term "Neuritin derivative" or "Neuritin variant" refers to a Neuritin polypeptide or Neuritin protein that 1) has been chemically modified, such as by the addition of polyethylene glycol or another compound, and / or 2) contains one or more substitutions of the nucleic acid or amino acid sequence, deletions, and / or insertions compared to Neuritin described in Figures 3 or 4. As used herein, the terms "biologically active polypeptide" and "biologically active fragment" are refer to a peptide or polypeptide having Neuritin activity, for example promote neurotogenesis in hippocampal or cortical neuronal cultures. As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount of Neuritin necessary to support one or more biological activities of Neuritin as described above. Neuritin polypeptides having use in the practice of the present invention may be full-length polypeptides of natural origin, or truncated polypeptides or peptides (eg, "fragments"). The polypeptides or fragments may be chemically modified, for example, glycosylated, phosphorylated and / or linked to a polymer, as described below, and these may have an amino terminus methionine, depending on how they are prepared. In addition, the polypeptides or fragments may be variants of the naturally occurring Neuritin polypeptide (eg, they may contain one or more deletions, insertions and / or amino acid substitutions as compared to naturally occurring Neuritin). The full-length Neuritin polypeptide or the fragment thereof can be prepared using the technology methods of Well-known recombinant DNA, such as those described in Sambrook et al.

(Mol e ul ar Cl oning: A Labora t ory Man ual, Cold Springer Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) and / or Ausubel et al., Eds.

. { Curren t Pro t ools in Mol ecul ar Bi olgy, Green Publishers Inc. and Wiley and Sons, NY [1994]). A gene or cDNA encoding the Neuritin protein or a fragment thereof can be obtained, for example, by selection of a genomic or cDNA library, or by PCR amplification. Alternatively, a gene encoding the Neuritin polypeptide or fragment can be prepared by chemical synthesis using methods well known to those of skill in the art, such as those described by Engels et al. (Angew. Chem. In tl. ., 28: 716-734 [1989]). These methods include, among others, the methods of phosphotries ter, phosphoramidite, and H-phosphonate for the synthesis of nucleic acid. A preferred method for such chemical synthesis is polymer-supported synthesis using the standard chemistry of phosphoramidite. Typically, the DNA encoding the Neuritin polypeptide will be several hundred nucleotides in length. Larger nucleic acids of about 100 nucleotides can be synthesized as several fragments using these methods. The fragments can then be ligated together to form the full-length Neuritin polypeptide. Usually, the DNA fragment encoding the amino terminus of the polypeptide will have an ATG, which codes for a methionine residue. This methionine may or may not be present on the mature form of the Neuritin polypeptide, depending on whether the polypeptide produced in the host cell is secreted from that cell.

In some cases, it may be desirable to prepare the nucleic acid and / or amino acid variants of the naturally occurring Neuritin. Nucleic acid variants (wherein one or more nucleotides are designed to differ from wild-type or naturally-occurring Neuritin) can be produced using site-directed mutagenesis or PCR amplification where the primer or primers have the desired point mutations ( see Sambrook et al., upra, and Ausubel et al., supra, for descriptions of mutagenesis techniques). The chemical synthesis using the methods described by Engels et al., Upra, can also be used to prepare such variants. Other methods known to those of skill in the art can also be used. Preferred nucleic acid variants are those that contain nucleic acid substitutions that explain the codon preference in the host cell that is to be used to produce Neuritin. Other preferred variants are those that code for conservative amino acid changes as described previously (for example, where the charge or polarity of the side chain of naturally occurring amino acids is not substantially altered by substitution with a different amino acid) compared to the wild type, and / or those designed either to generate a new site or sites of glycosylation and / or phosphorylation on Neuritin, or those designed to suppress one or more existing glycosylation and / or phosphorylation sites on Neuritin. The Neuritin gene or cDNA can be inserted into an expression vector suitable for expression in a host cell. The vector is selected to be functional in the particular host cell employed (eg, the vector is compatible with the machinery of the host cell, such that amplification of the Neuritin gene and / or gene expression can occur). The Neuritin polypeptide or the fragment thereof can be amplified / expressed in prokaryotic, yeast, insect host cells (Baculovirus systems) and / or eukaryotic cells.

The selection of the host cell will depend at least in part on whether the Neuritin polypeptide or the fragment thereof is to be glycosylated. If so, yeast, insect or mammalian host cells are preferable; the yeast cells will glycosylate the polypeptide, and the insect and mammalian cells can glycosylate and / or phosphorylate the polypeptide as it appears naturally on the Neuritin polypeptide (eg, "native" glycosylation and / or phosphorylation). Typically, the vectors used in any of the host cells will contain 5 'flanking sequences (also referred to as a "promoter") and other regulatory elements as well, such as one or more enhancers, an origin of the replication element, a transcriptional termination element. , a complete intron sequence containing a donor and acceptor splice site, a peptide signal sequence, an element of the ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide that is going to be expressed, and a selectable marker element. Each of these elements is discussed later. Optionally, the vector may contain a "tag" sequence, for example, an oligonucleotide sequence located at the 5 'or 3' end of the Neuritin coding sequence, which codes for polyHis (such as hexaHis) or another small immunogenic sequence. . This tag will be expressed together with the protein, and can serve as an affinity tag for the purification of the Neuritin polypeptide from the host cell. Optionally, the tag can be subsequently removed from the purified Neuritin polypeptide, by various means such as the use of a selected peptidase., for example. The 5 'flanking sequence can be homologous (e.g., from the same species and / or strain as the host cell), heterologous (e.g., from a different species of species or strain of the host cell), hybrid (e.g. example, a combination of 5 'flanking sequences from more than one source), synthetic, or it may be the 5' flanking sequence of Neuritin, native. As such, the source of the 5 'flanking sequence can be any unicellular prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, with the proviso that the 5' flanking sequence is functional in, and can be activated by, the machinery of the host cell. The 5 'flanking sequences useful in the vectors of this invention can be obtained by any of several methods well known in the art. Typically, the 5 'flanking sequences useful in the present different from the 5' flanking sequence of Neuritin, will have been previously identified by mapping and / or by restriction endonuclease digestion, and can thus be isolated from the appropriate tissue source using the appropriate restriction endonucleases. In some cases, the full length nucleotide sequence of the 5 'flanking sequence may be known. Here, the 5 'flanking sequence can be synthesized using the methods described above for the synthesis or cloning of nucleic acids. Where all or only a portion of the 5 'flanking sequence is known, it can be obtained using PCR and / or by selection of a suitable genomic library with oligonucleotide fragments and / or the 5' flanking sequence, originating therefrom or of another species. Where the 5 'flanking sequence is unknown, a DNA fragment containing a 5' flanking sequence can be isolated from a larger piece of DNA which may contain, for example, a coding sequence or even another gene or genes. Isolation can be accomplished by restriction endonuclease digestion using one or more carefully selected enzymes to isolate the appropriate DNA fragment. After digestion, the desired fragment can be isolated by purification on agarose gel, Quiagen® column or other methods known to the person skilled in the art. The selection of suitable enzymes to achieve this purpose will be readily apparent to one of ordinary skill in the art. The origin of the replication element is typically a part of the commercially acquired prokaryotic expression vectors, and aids in the amplification of the vector of a host cell. Amplification of the vector to a certain number of copies may, in some cases, be important for optimal expression of the Neuritin polypeptide. If the vector of choice does not contain an origin of the replication site, it can typically be synthesized based on a known sequence, and can be ligated into the vector. The transcription termination element is typically located 3 'to the end of the Neuritin polypeptide which codes for the sequence and serves to terminate the transcription of the Neuritin polypeptide. Usually, the termination element of the. Transcription in prokaryotic cells is a fragment rich in G-C followed by a poly-T sequence. While the element is easily cloned from a library or even commercially purchased as part of a vector, it can also be easily synthesized using methods for the synthesis of nucleic acids such as those described above. An element of the selectable marker gene encodes a protein necessary for the survival and growth of a host cell, grown in a selective culture medium. Selection marker genes, typical, encode for proteins that a) confer resistance to antibiotics or other toxins, for example, ampicillin, tetracycline, or kanamycin for prokaryotic host cells, b) complement the auxotrophic deficiencies of the cell; or c) provide critical nutrients not available from complex media. Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the racicline te resistance gene. The ribosome binding element, commonly referred to as the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes), is necessary for the initiation of mRNA translation. The element is typically located 3 'or downstream of the promoter, and 5' or upstream of the coding sequence of the Neuritin polypeptide to be synthesized. The Shine-Dalgarno sequence is varied but is typically a polypurine (for example, having a high content of A-G). Many Shine-Dalgarno sequences have been identified, each of which can be easily synthesized using methods described above and used in a prokaryotic vector. In those cases where it is desirable for Neuritin to be secreted from the host cell, a signal sequence can be used to direct the Neuritin polypeptide outside the host cell, where it is synthesized, and the carboxyl-terminal portion of the protein can be be suppressed in order to prevent membrane anchoring. Typically, the signal sequence is located in the coding region of the nucleic acid sequence of Neuritin, or directly from the 5 'end of the coding region of Neuritin. Many signal sequences have been identified, and any of them that are functional in the selected host cell can be used in conjunction with the Neuritin gene. Therefore, the signal sequence can be homologous or heterologous to the Neuritin polypeptide, and can be homologous or heterologous to the Neuritin polypeptide. In addition, the signal sequence can be chemically synthesized using the methods described above. In most cases, secretion of the polypeptide from the host cell via the presence of a signal peptide will result in the elimination of the amino-terminal methionine from the polypeptide. To facilitate secretion, the C-terminal region of the Neuritin polypeptide can be removed. This C-terminal region is approximately 27 amino acids in length, and many of the amino acids are hydrophobic; in addition, there is a consensual cleavage signal sequence which is found in many proteins anchored to glycosylphosphatidylinosi tol (GPI) in this region. In many cases, the transcription of the Neuritin polypeptide is increased by the presence of one or more introns on the vector; this is particularly true for eukaryotic host cells, especially mammalian host cells. The intron may be of natural origin within the nucleic acid sequence of Neuritin, especially where the Neuritin sequence used is a full length genomic sequence or a fragment thereof. Where the intron is not of natural origin, within the DNA sequence of Neuritin (as for most cDNAs), the introns can be obtained from another source. The position of the intron with respect to the 5 'flanking sequence and the coding sequence of Neuritin is important, since the intron must be transcribed to be effective. As such, where the nucleic acid sequence of Neuritin is a cDNA sequence, the preferred position for the intron is 3 'to the site of transcription initiation, and 5' for the polyA transcription termination sequence. Preferably for Neuritin cDNAs, the intron will be located on one side or the other (e.g., 5 'or 3') of the Neuritin coding sequence, such that it does not interrupt this coding sequence. Any intron from any source, including any viral, prokaryotic and eukaryotic organisms (plant or animal), can be used to practice this invention, provided that it is compatible with the host cell (s) into which it is inserted. Also included in this are the synthetic introns. Optionally, more than one intron can be used in the vector. Where one or more of the elements described above are not already present in the vector to be used, they can be individually obtained and ligated into the vector. The methods used to obtain each of the elements are well known to those skilled in the art and are comparable to the methods described above (for example, DNA synthesis, library selection, and the like). The final vectors used. to practice this invention are typically constructed from start vectors such as a commercially available vector. Such vectors may or may not contain some of the elements that are to be included in the finished vector. If none of the desired elements are present in the start vector, each element can be individually bound within the vector by cutting the vector with the appropriate restriction endonuclease (s), such that the ends of the element to be bound in and the ends of the vector are compatible for the ligature. In some cases, it may be necessary to "blunt" the ends that are to be joined together, in order to obtain a satisfactory ligation. Chroming is achieved primarily by filling the "sticky ends" using the Klenow DNA poly-erase or the T4 DNA polymerase in the presence of the four nucleotides. This procedure is well known in the art and is described for example in Sambrook et al., Supra. Alternatively, two or more of the elements to be inserted into the vector can be first linked together (if they are to be placed adjacent to each other) and then linked within the vector. Another method for constructing the vector is to conduct all the ligatures of the various elements simultaneously in a reaction mixture. Here, many meaningless or non-functional vectors will be generated, due to ligation or inadequate insertion of the elements, however, the functional vector can be identified and selected by restriction endonuclease digestion. Preferred vectors for the practice of this invention are those that are compatible with bacterial, insect and mammalian host cells. Such vectors include, among others, pCRII (Invitrogen Company, San Diego, CA), pBSII (Stratagene Company, La Jolla, CA), and pETL (BlueBacII; Invitrogen). After the vector has been constructed and a Neuritin nucleic acid has been inserted into the appropriate site of the vector, the terminated vector can be inserted into a suitable host cell for amplification and / or expression of the Neuritin polypeptide. The host cells can be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell or a vertebrate cell). The host cell, when cultured under appropriate conditions, can synthesize Neuritin protein which can be subsequently collected from the culture medium. (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). After harvesting, the Neuritin protein can be purified using methods such as molecular sieve chromatography, affinity chromatography, and the like. The selection of the host cell will depend in part on whether the Neuritin protein is going to be glycosylated or phosphorylated (in which case eukaryotic host cells are preferred), and the manner in which the host cell is able to "fold" the protein to its native tertiary structure (eg, the proper orientation of the disulfide bridges, etc.) such that the biologically active protein is prepared by the cell. However, where the host cell does not synthesize biologically active Neuritin, Neuritin can be "folded" after synthesis using appropriate chemical conditions, as discussed below. Suitable cells or cell lines can be mammalian cells, such as Chinese hamster ovary cells (CHO) or 3T3 cells. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, selection and production of the product and purification thereof are known in the art. Other suitable mammalian cell lines are the COS-1 and COS-7 monkey cell lines, and the CV-1 cell line. In addition, exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from the culture within the primary tissue, as well as primary explants, are also suitable. The candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene. Other suitable mammalian cell lines include, but are not limited to, HeLa cells, mouse L-929, 3T3 lines derived from Swiss, Balb-c or NIH mice, hamster BHK or HaK cell lines. Similarly, useful as host cells suitable for the present invention are bacterial cells. For example, the various strains of E. col i (for example, HB101, DH5a, DH10, and MC1061) are well known as host cells in the field of biotechnology. Different strains of B can also be used in this method. s ubti l i s, Pseudomonas spp. , others Ba cil l us spp, S t rep tomyces spp. , and similar. Many strains of yeast cells known to those of skill in the art are also available as host cells for the expression of the polypeptides of the present invention. In addition, where desired, the insect cells can be used as host cells in the method of the present invention (Miller et al., Gen e ti c Engin eering 8: 277-298 [1986]). The insertion (also referred to as "transformation" or "transfection") of the vector within the selected host cell can be achieved using methods such as electroporation, microinjection, lipofection with calcium chloride or the DEAE-dextran method. The selected method will be in part a function of the type of host cell that will be used. These methods and other suitable methods are well known to those of skill in the art, and are described for example in Sambrook et al., Supra. Host cells that contain the vector (eg, transformed or transfected) can be cultured using standard means well known to the person skilled in the art. The media will usually contain all the nutrients necessary for the development of cell survival. Suitable media for the cultivation of E cells. col i are for example, Luria Broth (LB) and / or Terrific Broth (TB). Suitable means for culturing eukaryotic cells are RPMI 1640, MEM, DMEM, all of which can be supplemented with serum and / or growth factors, as required by the particular cell line being cultured. A suitable medium for insect cultures is Grace's medium supplemented with levadurolate, lactalbumin hydrolyzate, and / or fetal calf serum as necessary. Typically, an antibiotic or other compound useful for the selective development of the transformed cells is added as a supplement to the media. The compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed. For example, where the selectable marker element is the resistance to kanamycin, the compound added to the culture medium will be kanamycin. The amount of Neuritin polypeptide produced in the host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, high resolution liquid chromatography (HPLC) separation, immunoprecipitation, and / or activity assays such as assays. of displacement of gel binding to DNA. If the Neuritin polypeptide has been designed to be secreted from the host cell, most of the polypeptide will likely be found in the cell culture medium. Polypeptides prepared in this manner will typically not possess a methionine at the amino terminus, since it is removed during secretion from the cell. However, if the Neuritin polypeptide is not secreted from host cells, it will be present in the cytoplasm (for gram-positive eukaryotic bacteria, and insect host cells) or in the periplasm (from host cells of gram-negative bacteria). ) and may have a methionine at the amino terminus. For the intracellular Neuritin protein, the host cells are typically first mechanically or osmotically broken to release the cytoplasmic content towards a buffered solution. The Neuritin polypeptide can then be isolated from this solution. Purification of Neuritin polypeptide from the solution can be achieved using a variety of techniques. If the polypeptide has been synthesized such that it contains a label such as Hexahistidine (Neuri tina / hexaHis) or another small peptide either at its carboxyl or amino terminus, it can be essentially purified in a one-step process, by passing of the solution through an affinity column where the column matrix has a high affinity for the tag and the polypeptide directly (for example, a monoclonal antibody that specifically recognizes Neuritin). For example, polyhistidine binds with great affinity and specificity to nickel, thus a nickel affinity column (such as Qiagen nickel columns) can be used for the purification of Neuri tina / polyHis. (See for example, Ausubel et al., Eds., Curren t Pro t ools in Mol ecul ar Bi olgy, Section 10.11.8, John Wiley & amp;; Sons, New York [1993]). Where the Neuritin polypeptide is unlabeled and antibodies are not available, other well-known methods for purification can be used. Such methods include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and comparative isoelectric focusing (machine / "Isoprime" technique, Hoefer Scientific). In some cases, two or more of these techniques can be combined to achieve increased purity. Preferred methods for purification include labeling with poliHis t idine and ion exchange chromatography, in combination with the preparative isoelectric focus. If it is anticipated that the polypeptide Neuritin will be found mainly in the periplasmic space of the bacteria or the cytoplasm of eukaryotic cells, the content of the periplasm or cytoplasm, including the inclusion antibodies (eg, gram-negative bacteria) if the processed polypeptide has formed such complexes, it can be extracted from the host cell using any standard technique known to the person skilled in the art. For example, the host cells can be lysed to release the periplasm content by French press, homogenization, and / or sonication. The homogenate can then be centrifuged. If the Neuritin polypeptide has formed inclusion bodies in the periplasm, the inclusion bodies can often bind to the internal and / or external cell membranes, and thus will be found mainly in the concentrate or button material after centrifugation. The button material can then be treated with a chaotropic agent such as guanidine or urea to liberate, break, and solubilize the inclusion bodies. The Neuritin polypeptide in its now soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If desired, to isolate the Neuritin polypeptide, the isolation can be carried out using standard methods such as those described below and in Marston et al. (Me., In z., 182: 264-275 [1990]). If the inclusion bodies of the Neuritin polypeptide are not formed to a significant degree in the periplasm of the host cell, the Neuritin polypeptide will be found mainly in the supernatant after the centrifugation of the cell homogenate, and the Neuritin polypeptide can be isolated of the supernatant using methods such as those described below. In those situations where it is preferable to partially or completely isolate the Neuritin polypeptide, the purification can be carried out using standard methods well known to the person skilled in the art. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieves, and / or ion exchange), and / or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods to complete the purification. In addition to preparing and purifying the Neuritin polypeptide using recombinant DNA techniques, the polypeptides, fragments and / or Neuritin derivatives can be prepared by chemical synthesis methods (such as solid phase peptide synthesis) using methods known in the art. such as those described by Merrifield et al. (J. Am. Ch., Soc., 85: 2149 [1964]), Houghten et al. (Proc.Nat.Acid.Sci. USA, 82: 5132 [1985]) , and Stewart and Young (Solid Phase Peptide Synthesis, Pierce Chem. Co., Rockford, IL [1984]). Such polypeptides can be synthesized with or without a methionine on the amino terminus. The chemically synthesized Neuritin polypeptides or fragments thereof can be oxidized using the methods described in these references to form disulfide bridges. Neuritin polypeptides or fragments thereof can be used as biologically active or immunological substitutes for purified Neuritin polypeptides, naturally occurring in the therapeutic and immunological processes. The chemically modified Neuritin compositions (eg, "derivatives") wherein the Neuritin polypeptide is linked to a polymer ("Neurine tina-polymers") are included within the scope of the present invention. The selected polymer is typically soluble in water, such that the protein to which it binds does not precipitate in an aqueous environment, such as a physiological environment. The selected polymer is usually modified to have a simple reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled as provided in the present methods. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or the mono-alkoxy or aryloxy derivatives of 1 to 10 carbon atoms thereof (see US Pat. No. 5,252,714). The polymer can be branched or unbranched. Included within the scope of the Neuritin polymers is a mixture of polymers. Preferably, for the therapeutic use of the preparation of the final product, the polymer will be pharmaceutically acceptable. The water soluble polymer or mixture thereof may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxy polyol glycol, dextran, cellulose, or other polymers based on carbohydrate, poly- (N-vinylpyrrolidone) -polyethylene glycol , propylene glycol homopolymers, a copolymer of polypropylene oxide / ethylene oxide, polyoxyethylated polyols (eg, glycerol) and polyvinyl alcohol. For the acylation reactions, the selected polymer (s) must have a simple reactive ester group. For reductive alkylation, the selected polymer (s) must have a reactive, simple aldehyde group. The polymer may be of any molecular weight, and may be branched or unbranched. The pegylation of Neuritin can be carried out by any of the pegylation reactions known in the art, as described for example in the following references: Focus on Growth Fa c t ors 3: 4-10 (1992); European Patent EP-0, 154, 316; and European Patent EP-0,401,384. Preferably, the pegylation is carried out by means of an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or a water soluble polymer, reagent, analog) as described below. PEGylation by acylation generally involves the reaction of an active ester derivative of polyethylene glycol (PEG) with a Neuritin protein. Any reactive PEG molecule, known or subsequently discovered, can be used to carry out the pegylation of Neuritin. An activated, preferred PEG ester is PEG esterified to N-hydroxysuccinimide ("NHS"). As used herein, "acylation" is contemplated to include, without limitation, the following types of linkages between Neuritin and a water soluble polymer such as PEG: amide, carbamate, urethane, and the like, as described in Bi oconj uga te Ch em. 5: 133-140 (1994). The reaction conditions can be selected from any of those known in the pegylation art or those subsequently developed, provided conditions such as temperature, solvents, and pH that could inactivate the Neuritin species that is going to be inactivated are avoided. modified. PEGylation by acylation usually results in a polypeglylated Neuritin product, wherein the e-amino groups of the lysine are pegylated via an acyl linkage group. Preferably, the connection link will be an amide. Also preferably, the resulting product will be at least about 95 percent mono-, di- or tri-pegylated. However, some species with higher degrees of PEGylation (up to the maximum number of e-amino acid groups of Neuritin lysine plus an a-amino group at the amino terminus of Neuritin) will normally be formed in amounts dependent on the specific reaction conditions used. If desired, more pegylated, purified species can be separated from the mixture, particularly unreacted species, by standard purification techniques, including, among others, dialysis, salification, ultrafiltration, ion exchange chromatography, gel filtration chromatography and electrophoresis Pegylation by alkylation generally involves the reaction of a terminal aldehyde derivative of the PEG with a protein such as Neuritin in the presence of a reducing agent. Regardless of the degree of PEGylation, the PEG groups are preferably linked to the protein by means of a group -CH ^ -NH-. With particular reference to the -CH2- group, this type of bond is referred to herein as an "alkyl" bond. Derivatization by means of reductive alkylation to produce a single-sided product exploits the differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in Neuritin. Typically, the reaction is performed at a pH (see below) which allows to take advantage of pKa differences between the e-amino groups of the lysine residues and the a-amino group of the N-terminal residue of the protein. By such selective derivatization, the coupling of a water-soluble polymer containing a reactive group, such as an aldehyde, to a protein is controlled: the conjugation with the polymer occurs predominantly at the N-terminus of the protein, without significant modification of other reactive groups such as the side chain amino groups of lysine. The present invention provides a substantially homogeneous preparation of Neuri tina-monopolymer protein conjugated molecules (which means that the Neuritin protein to which the polymer molecule has been substantially coupled only (eg, at least about 95%) in a single site on the Neuritin protein). More specifically, if polyethylene glycol is used, the present invention also provides the pegylated Neuritin protein which possibly lacks antigenic linking groups, and which has the polyethylene glycol molecule directly coupled to the Neuritin protein. A particularly preferred water-soluble polymer for use herein is polyethylene glycol, abbreviated as PEG. As used herein, polyethylene glycol is understood to encompass any of the PEG forms that have been used to derivatize other proteins, such as mono- (C 1 -C 10 alkoxy) or (aryloxy) -polyethylene glycol. In general, chemical derivatization can be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule. The methods for preparing the pegylated neuron will generally comprise the steps of a) reacting a Neuritin polypeptide with polyethylene glycol (such as a reactive ester or PEG aldehyde derivative) under conditions under which Neuritin becomes bound to one or more PEG groups; and b) obtaining the reaction product (s). In general, the optimum reaction conditions for the acylation reactions will be determined based on the known parameters and the desired result.

For example, the higher the PEG: protein ratio, the higher the percentage of poly-pegylated product. Reductive alkylation to produce a substantially homogeneous population of conjugated mono-polymer molecule / Neuritin protein will generally comprise the steps of: a) reacting a Neuritin protein with a reactive PEG molecule under reductive alkylation conditions, at a pH suitable for allow selective modification of the a-amino group at the amino terminus of the Neuritin protein; and b) obtaining the reaction product (s). For a substantially homogeneous population of mono-polymer / Neuritin protein conjugated molecules, the reaction conditions of the reductive alkylation are those that allow selective coupling of the water-soluble polymer portion to the N-terminus of Neuritin. Such reaction conditions generally give pKa differences between the amino groups of lysine and the a-amino group at the N-terminus (pKa being the pH at which 50% of the amino groups are protonated and 50% are not) . The pH also affects the ratio of the polymer to the protein to be used. In general, if the pH is lower, an excess of polymer will be desired to the protein (for example, the less reactive the N-terminal α-amino group, the more polymer will be needed to achieve optimum conditions). If the pH is higher, the polymer: protein ratio does not need to be that large (for example, the more reactive groups are available, the fewer polymer molecules are needed). For purposes of the present invention the pH will generally fall within the range of 3 to 9, preferably 3 to 6. Another important consideration is the molecular weight of the polymer. In general, the higher the molecular weight of the polymer, the lower the number of polymer molecules that can be coupled to the protein. Similarly, the branching of the polymer must be taken into account when optimizing these parameters. In general, the higher the molecular weight (or the more branches there are), the higher the polymer: protein ratio. In general, for the pegylation reactions contemplated herein, the preferred average molecular weight is from about 2 kDa to about 100 kDa (the term "about" indicates ± 1 kDa). The preferred average molecular weight is about 5 kDa to about 50 kDa, particularly preferably about 12 kDa to about 25 kDa. The ratio of water-soluble polymer to the protein Neuritin will generally be in the range of 1: 1 to 100: 1, preferably (for pegylation) 1: 1 to 20: 1 and (for monopegylation) 1: 1 to 5 :1. Using the conditions indicated above, the reductive alkylation will provide selective coupling of the polymer to any Neuritin protein having an a-amino group at the amino terminus, and will provide a substantially homogeneous preparation of the monopolymer / protein Neuritin conjugate. The term "monopolymer / protein Neuritin conjugate" is used herein to mean a composition comprised of a simple polymer molecule bound to a protein molecule Neuritin. The monopolymer / protein Neuritin conjugate will preferably have a polymer molecule located at the N-terminus, but not on the amino-side groups of the lysine. The preparation will preferably be greater than 90% monopolymer / protein Neuritin conjugate, and more preferably greater than 95% monopolymer / Neuritin protein conjugate, with the rest of the observable molecules remaining unreacted (for example, the protein lacking the polymer portion). The following examples provide a preparation that is at least about 90% monopolymer conjugate / protein, and about 10% unreacted protein. The monopolymer / protein conjugate has biological activity. For the present reductive alkylation, the reducing agent must be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. Preferred reducing agents can be selected from the group consisting of sodium borohydride, sodium cyanoborohydride, dimethylaminoborane, tri-ethylaminoborane and pyridinborane. A particularly preferred reducing agent is sodium cyanoborohydride. Other reaction parameters, such as the solvent, the reaction times, the temperatures, etc., and the means of purification of the products, can be determined based on the published information regarding the derivatization of proteins with soluble polymers in Water. A mixture of polymer-protein Neuritin conjugate molecules can be prepared by acylation and / or alkylation methods, as described above, and the proportion of monopolymer / protein conjugate can be selected to be included in the mixture. Thus, where desired, a mixture of various proteins can be prepared with various numbers of bound polymeric molecules (eg, di-, tri-, tetra-, etc.) and combined with the monopolymer / protein conjugate material Neuritin. using the present methods. In general, conditions that can be alleviated or formulated by administration of the present polymer / Neuri t ina, include those described herein for Neuritin molecules in general. However, the polymer / Neuritin molecules described herein may have additional activities, improved or reduced activities, or other characteristics, as compared to non-derivatized molecules. Neuritin nucleic acid molecules, fragments, and / or derivatives that do not themselves encode the polypeptides that are active in the activity assays, can be useful as hybridization probes in diagnostic assays to be tested, either qualitatively or quantitatively , the presence of DNA or Neuritin RNA in mammalian tissue or in body fluid samples. Fragments and / or derivatives of the Neuritin polypeptide that are not themselves active in activity assays, may be useful as modulators (eg, inhibitors or stimulators) of Neuritin receptors in vi t ro oin vi vo, or for prepare antibodies for the Neuritin polypeptides. Neuritin polypeptides and fragments thereof, whether or not they are chemically modified, can be used alone, or in combination with other pharmaceutical compositions such as, for example, neurotrophic factors, cytokines, rferons, rleukins, growth factors , antibiotics, anti-inflammatories, agonists or antagonists of neurotransmitter receptors and / or antibodies, in the treatment of disorders of the neurological system. Neuritin polypeptides and / or fragments thereof can be used to prepare antibodies generated by standard methods. Thus, antibodies that react with Neuritin polypeptides, as well as reactive fragments of such antibodies, are also contemplated within the scope of the present invention. The antibodies can be polyclonal, monoclonal, recombinant, chimeric, single chain and / or bispecific. Typically, the antibody or fragment thereof will be "humanized", for example, prepared to prevent or minimize an immune reaction to the antibody, when administered to a patient. The antibody fragment can be any fragment that is reactive with the Neuritin of the present invention, such as Fab, Fab ', etc. Also provided by this invention are hybridomas generated by the presentation of Neuritin or a derivative thereof as an antigen, to a selected mammal, followed by fusion of the cells (eg, spleen cells) of the animal with certain cells. cancer cells to create immortalized cell lines, by known techniques. The methods employed to generate such cell lines and the antibodies directed against all or portions of a human Neuritin polypeptide of the present invention are also encompassed by this invention. The antibodies can be used therapeutically, such as to inhibit the binding of Neuritin to its receptor. The antibodies can also be used for diagnostic purposes in vi and vi vi, such as in the marked form to detect the presence of Neuritin in a body fluid.

Therapeutic Compositions and Administration Therapeutic compositions for treating the various disorders of the neurological system are within the scope of the present invention. Such compositions may comprise a therapeutically effective amount of a Neuritin polypeptide or fragment thereof (any of which may be chemically modified) in admixture with a pharmaceutically acceptable carrier. The carrier material can be water for injection, preferably supplemented with other common materials in solutions for administration to mammals. Typically, a Neuritin therapeutic compound will be administered in the form of a composition comprising the purified protein (which may be chemically modified) in conjunction with one or more physiologically acceptable carriers, excipients, or diluents. Saline solution or neutral buffered saline, mixed with serum albumin are suitable exemplary carriers. Preferably, the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose). Other carriers, diluents, and standard excipients may be included as desired. Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0 to 5.5, which may also include sorbitol or a suitable substitute therefor. The Neuritin compositions can be systematically administered parenterally.

Alternatively, the compositions may be administered intravenously or subcutaneously. When administered systemically, the therapeutic compositions for use in this invention may be in the form of a parenterally acceptable, pyrogen-free aqueous solution. The preparation of such pharmaceutically acceptable protein solutions, with respect to pH, isotonicity, stability and the like, is within the skill in the art. Therapeutic formulations of the Neuritin compositions useful for practicing the present invention can be prepared for storage by mixing the selected composition having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remi ngt on 's). Ph a rma ce u ti ca l Sci en ces, 18th edition, AR Gennaro, ed-, Mack Publishing Company [1990]) in the form of a lyophilized cake or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to patients and are preferably inert at the doses and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronics or polyethylene glycol (PEG). The composition of Neuritin that is to be used for the administration should not be sterile. This is easily achieved by filtration through sterile filtration membranes. Where the Neuritin composition is lyophilized, sterilization using these methods can be conducted either before, or after lyophilization and reconstitution. The composition for parenteral administration will ordinarily be stored in lyophilized form or in solution.

Therapeutic compositions are generally placed in a container having a sterile access port, for example, a bag for intravenous solution or a bottle having a plug pierceable by a hypodermic injection needle. The route of administration of the composition is in accordance with known methods, for example, oral, injection or infusion routes by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, or intralesional route, or by sustained release systems or implantation devices that may involve optionally the use of a catheter. Where desired, the compositions can be administered continuously by infusion, bolus injection or by implantation device. Alternatively or additionally, Neuritin can be administered locally by implantation in the affected area of a membrane, sponge, or other appropriate material upon which the Neuritin polypeptide has been absorbed.

Where an implantation device is used, the device can be implanted into any suitable tissue or organ, such as, for example, within a cerebral ventricle or in the brain parenchyma, and the distribution of Neuritin can be directly through the brain. device by means of a bolus or continuous administration, or by means of a catheter using continuous infusion. The Neuritin polypeptide can be administered in a sustained release formulation or preparation. Suitable examples of sustained release preparations include semipermeable polymer matrices in the form of shaped articles, eg, films, or microcapsules. Sustained-release matrices include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919, European Patent EP-58,881), copolymers of L-glutamic acid and gamma-yl-L-glutamate (Sidman et al. Bi opolymers, • 22: 547-556 [1983]), poly- (2-hydroxyethyl-methacrylate) (Langer et al., J. Bi omed, Ma t er. Res., 15: 167-277] 1981] and Langer, Chem. Tech., 12: 98-105 [1982]), ethylene vinyl acetate (Langer et al., Supra) or poly-D (-) - 3-hydroxybutyric acid (European Patent EP-133,988). Sustained-release compositions can also include liposomes, which can be prepared by any of the various methods known in the art (eg, German Patent DE-3, 218, 121; Epstein et al., Proc. Na ti. Acad. Sci. USA, 82: 3688-3692 [1985]; Hwang et al., Proc. Na ti. Acad. Sci. USA, 77: 4030-4034 [1980]; European Patents EP-52,322; EP-36,676; EP- 88,046; EP-143, 949). In some cases, it may be desirable to use the Neuritin compositions in an ex vivo manner, for example, to treat cells or tissues that have been removed from the patient and are subsequently subsequently implanted into the patient. In other cases, Neuritin can be distributed through implantation in patients of certain cells that have been genetically engineered (using the methods described above) to express and secrete the Neuritin polypeptide. Such cells can be human cells, and can be derived from the patient's own tissue or from another source, be it human or non-human. Optionally, the cells can be immortalized. The cells can be implanted inside the brain, the adrenal glands or in other body tissues or organs. In certain situations, it may be desirable to use gene therapy methods for the administration of Neuritin to patients suffering from certain neurological disorders. In these situations, the genomic DNA, the cDNA, and / or the synthetic DNA encoding Neuritin or a fragment or variant thereof, can be operatively linked to a constitutive or inducible promoter that is active in the tissue within which the composition will be injected. This construction of Neuritin DNA, either inserted into a vector, or only without a vector, can be injected directly into the brain or into another tissue, whether neuronal or non-neuronal. Alternatively, a Neuritin DNA construct can be directly injected into the muscle tissue where it can be collected into the cells and expressed in the cells, provided that the Neuritin DNA is operably linked to a promoter that is active in the muscle tissue, such as the cytomegalovirus (CMV) promoter, the Rous sarcoma virus (RSV) promoter, or the muscle creatine kinase promoter. Typically, the DNA construct can include (in addition to the Neuritin DNA and a promoter), the vector sequence obtained from the vectors such as the adenovirus vector, adeno-associated viral vector, a retroviral vector, and / or a vector of the herpes virus. The vector / DNA construct can be mixed with one or more pharmaceutically acceptable carriers, for injection. An effective amount of the Neuritin composition (s) to be employed therapeutically will depend, for example, on the therapeutic objectives such as the indication for which the .Neuritin is to be used, the route of administration, and the condition of the patient. Consequently, it will be necessary for the therapist to titrate the dose and modify the administration route as required, in order to obtain the optimal therapeutic effect. A typical daily dose may be in the range of about 0.1 μg / kg to about 100 mg / kg or more, depending on the factors mentioned above. Typically, a clinician will administer the Neuritin composition until a dose is achieved that achieves the desired effect. The Neuritin composition can therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of Neuritin) over time, or as a continuous infusion by means of implantation device or catheter. As additional studies are conducted, information will emerge regarding the appropriate dose levels for the treatment of various conditions in various patients, and the worker of ordinary experience, considering the therapeutic context, the type of disorder under treatment, age and general health. of the patient, will be able to assess the appropriate dose. In general, the dose will be between 0.01 μg / kg of body weight (calculating the mass of the protein alone, without chemical modification) and 300 μg / kg (based on them). Neuritin proteins, fragments and / or derivatives thereof can be used to treat diseases and disorders of the central or peripheral nervous system, which may be associated with alterations in the expression pattern of Neuritin, or which may benefit of exposure to Neuritin or anti-Neuritin antibodies. The Neuritin protein and / or the fragments or derivatives thereof, can be used to treat patients in whom the various cells of the central, autonomic or peripheral nervous system have degenerated and / or have been damaged by congenital disease, trauma, mechanical damage, surgery, attack, ischemia, infection, metabolic disease, nutritional deficiency, malignancy, and / or toxic agents. More specifically, Neuritin protein levels can be modulated (sub or supraregulated) for indications such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, Charcot-Marie-Tooth syndrome, Huntington's disease, peripheral neuropathy induced by diabetes or other metabolic disorder, and / or dystrophies or degeneration of the neural retina, such as retinitis pigmentosa, drug-induced retinopathies, stationary forms of night blindness, progressive degeneration of cones and rods, and the like. In other embodiments of the present invention, the Neuritin protein or polypeptide or fragments or derivatives thereof, may be used in conjunction with surgical tissue implantation in the treatment of diseases in which the tissue implant is indicated.

DNA deposit The cells of E. col i containing the pCRScript SK + plasmid into which the cDNA encoding full-length human neurotin (1-142 amino acids) has been inserted, has been deposited with the ATCC North American Species Crop Collection (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA) on August 9, 1996 as accession number 98134. The following examples are intended to be for illustration purposes only, and should not be considered as limiting the scope of the invention, of any mode.

EXAMPLES Example I: Cloning of Neuritin cDNA Male rats (Wistar) of approximately 8 to 10 weeks of age (with approximately 230 to 300 grams of body weight) were injected intraperitoneally with approximately 8 mg / kg of body weight of kainate (prepared in a stock solution of 5%). mg / ml kainate in phosphate buffered saline [PBS]) Approximately six hours later, the animals were sacrificed, and the region of the dentate gyrus gyrus of the brain was removed and stored in liquid nitrogen. The DG tissue of approximately 100 animals was combined, and the RNA from this tissue was prepared by a modification of the guanidinium thiocyanate method ("GTC", Chomczynski et al., Anal. Bi ochem., 162: 156 [1987] ). After lysis of the tissue in GTC, 2 extractions with phenol were performed, followed by an extraction with chloroform, and the RNA was precipitated and resuspended in water. Poly (A) + RNA was selected using the oligo- (dT) -cellulose columns (Clontech, Palo Alto, CA). This RNA (and the corresponding cDNA) is referred to herein as "activated DG" RNA or cDNA. For the analysis of subtraction, for the construction of the library and the cDNA probe (all of which are described below), the RNA was treated with DNase (RNAse free; Promega, Madison, Wisconsin) to remove any contaminating genomic DNA. The same protocol described above was used to prepare the poly (A) + RNA from the normal dentate gyrus tissue, and the total brain tissue from male rats of the same age that were not treated with kainate. The first cDNA letter was synthesized in two 50 μl reactions using poly (A) + DG-activated RNA prepared as described above. Each reaction contained approximately 5 μg of RNA in approximately 30 μl of reverse transcriptase buffer (Gubler et al., Gen e, 25: 263-269 [1983]), approximately 1 μl of RNase (4 μ / μl; Promega, Madison, Wl), approximately 1 μg of oligo- (dT) -Xba l primer adapter (Promega, Madison, Wisconsin), approximately 30 μCi of 32P-dCTP (approximately 3,000 Ci / mol, Amersham, Arlington Heights, IL) and approximately 400 μg of cloned reverse transcriptase MLV (BRL, Grand Island, NY). After about 60 minutes at about 37 ° C, the RNA was hydrolyzed for about 20 minutes at about 68 ° C by the addition of about 10 μl of sodium hydroxide (1 N), about 2 μl of EDTA (0.5 M) and water to approximately 100 μl. The RNA was then placed on ice, and then neutralized with approximately 10 μl of 1 M HCl. Approximately 5 μg of transfer RNA was added, and the mixture was centrifuged through a rotating Sephadex G-50 column. The cDNA recovery was determined by comparing the radioactivity in the column eluate to that in the sample originally applied to the column. The two cDNA samples were combined, the ethanol was precipitated with ammonium acetate, and resuspended to approximately 10 ng / μl in water. The cDNA was mixed with an equal volume of poly (A) + RNA from rat brain (1 μg / μl) previously coupled to biotin using two rounds of photobiotinylation (Clontech, Palo Alto, CA; see Sive et al., Nucl ei c Aci ds Res, 16: 10937 [1988]) and then ethanol precipitated with ammonium acetate. After resuspension to approximately 100 ng / μl of cDNA and approximately 10 μg / μl of RNA in formamide buffer (40% formamide, 50 mM Hepes, pH 7.6, 0.5 M sodium chloride, 2 mM EDTA), the solution it was placed in glass capillaries (25 μl each) that were sealed. The capillaries were incubated approximately 3 minutes at 68 ° C and then for two days at 52 ° C. The capillaries were broken to open and the contents of each were added to approximately 180 μl of buffer (Hepes pH 7.6 50 M, 0.5 M NaCl, 2 mM EDTA). Streptavidin (Vector Labs, Burlingame, CA) was added at approximately 1 μg per μl of biotinylated RNA, and the mixtures were incubated for approximately 10 minutes at room temperature. After incubation, two extractions were conducted with phenol / chloroform (1 volume: 1 volume) and one extraction with chloroform. The recovered aqueous phase typically contained 10 to 20% of the total cDNA used for cloning by subtraction. This single-stranded cDNA was precipitated with ethanol and resuspended in approximately 16 μl of water. About 1 μl of dATP (10 mM) and about 4 μl of 5 X TdT buffer (Boehringer Manheim) were added to the cDNA. After about 3 minutes at 100 ° C and cooling on ice, the terminal deoxynucleot idyl transferase (17 μl, Boehringer, Manheim, Germany) was added and the mixture was incubated for about 2 hours at about 37 ° C. Two micrograms of oligo- (dT) -Xba l (Promega, Madison, Wl) were added as the primer adapter, and the mixture was incubated approximately 5 minutes at 60 ° C. Analysis of the second strand was conducted in approximately 50 μl of total volume containing 90 mM Hepes buffer pH 6.6, 10 mM magnesium chloride, the 4 deoxynucleotide triphosphates at a concentration of approximately 0.5 M each, approximately 10 mM DTT , and 10 Klenow units (Boehringer Manheim sequencing grade). After approximately 6 hours at room temperature, another aliquot of the enzyme was added, and the mixture was incubated for an additional period of three hours. The reaction was stopped with extractions of phenol and chloroform, after which 5 μg of transfer RNA was added, and the cDNA was precipitated with ethanol and with ammonium acetate. The double-stranded cDNA was resuspended in approximately 9.6 μl of water and digested for 5 hours at 37 ° C with 10 units of the restriction enzyme Xba I (Boehringer), after which it was loaded onto a thin agarose gel at 100 ° C. 1% and underwent electrophoresis. The cDNA molecules containing the gel splice, greater than about 550 pairs of passages ("bp") in size, were excised, the cDNA was extracted using QIAEX (Qiagen Corp., Chatsworth, CA) and recovery (approximately 10 % of the total subtracted cDNA) was determined by radioactive counting. This cDNA was ligated into the arms of the lambda-ZAP vector (Stratagene, La Jolla, CA) that were previously digested with Xba I, and treated with Calf Intestinal Phosphatase (Boehringer Manheim). The ligatures were conducted using approximately 1 μl of phage arms and various concentrations of cDNA (3-20 ng). The ligatures were packaged (Gigapack, Stratagene, La Jolla, CA) the phage titer was determined. The library was plated at low density, individual plates were collected separately, and the plasmids in the pBluescript vector were removed from the phage following the manufacturer's protocol (see Short et al., Nu cl ei c Aci ds Res. 1_6: 7583- 7600 [1988]). The plasmid DNA was prepared from E cells. col i previously transformed with pBluescript plasmids using standard minipreparation procedures (see Sambrook et al., Mol ecu ar Cloning: A Labora t ory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]). Approximately two to four μg of plasmid DNA obtained from the minipreparation were digested with Xba I and Southern blot analysis was performed on the Hybond N + filters (Amersham, Arlington Heights, IL) using standard procedures. To prepare the probes to select the filters containing the cDNA, approximately 100 ng of the single-stranded cDNA of each type (control and activated by DG) was radiolabeled by adding it to a mixture containing approximately 12 μl of random primers ( Boehringer, approximately 90 26oU / ml), approximately 2 MCi of 3IP-dCTP (3,000 Ci / mmol; Amersham, Arlington Heights, IL), approximately 0.6 mM of each dATP, dTTP, and dGTP and 40 μL of Klenow (Boehringer, approximately 2 U / μL) were used in 800 μL of the buffer, for the second synthesis of the cDNA strand (see above). The incubation was conducted overnight at room temperature in 2 aliquots of 400 μl. This reaction resulted in probes of approximately 1.2 x 109 cpm. After at least 6 hours of prehybridization at about 42 ° C in hybridization buffer (see below), the cDNA spots were hybridized to the probes. A spot hybridized to the activated DG probes, and a duplicate spot hybridized to the control probes (normal DG cDNA). Hybridizations were performed in a solution containing 50% formamide, 5X SSCPE (Sambrook et al., 1989), 10 X Denhardt, 0.5% SDS, 0.5 mg / ml herring sperm DNA and the cDNA probe a a concentration of approximately 1 x 10d cpm / ml. The stains were incubated for approximately 48 hours in a water bath with stirring at 42 ° C. After incubation, the spots were washed with 0.1 X SSC, 0.2% SDS, 3 times for 1 hour each at 68 ° C. After exposure to the film, those cDNA clones that hybridize stronger to the activated DG probes than the control probes were selected and reselected using a second group of activated DG and control probes prepared as described above . Those clones that hybridized more strongly to the activated DG probe than to the control probe in the two separate selections, were sequenced from both ends (approximately 200 to 300 base pairs from each end) using the standard sequencing methods, and these sequences were searched by FASTA analysis (Pearson et al., Proc. Na ti, Acad.Sci. USA, 85, 2444-2448 [1988]) in the GenBank DNA databases and other public databases. Based on the sequential comparison, several clones appeared to be novel. The clones that relate to the present invention were designed as follows: # 784, # 1441, # 2090; # 2268; # 2282; # 7547; # 8032; # 6734; and # 7761. To obtain the full-length cDNA clones, a second cDNA library was constructed from activated DG RNA, using methods similar to those described above. The library was made as described above using the activated DG poly-A RNA and the oligo (dT) -Xba l (Promega) primers, but only the larger 1.5 kb cDNAs were selected as inserts. This library was plated at high density and transferred to nylon filters (S &S, Keene, NH). A probe of the Xba l insert of approximately 0.5 kbp of clone # 1441 was generated by isolation of this fragment restricted with Xba I using the Qiagen Purification Kit.

(Qiagen, Chatsworth, CA) and following the manufacturer's recommendations. The fragment was then radiolabelled with a-J2P-dCTP using standard methods (RediVue, Amersham, Arlington Heights, IL). The filters were hybridized using the conditions as described above. Several positive clones were identified from this selection. Two of the positive clones, 1441-10 and 1441-13, were selected since they had the longest inserts (approximately 1.6 kbp and 1.4 kbp, respectively). These two clones were subjected to DNA sequence analysis on both strands using the method of dideoxy chain determination with fluorescent dideoxynucleotides (Applied Biosystems Inc., Foster City, CA). The nucleotide sequence was analyzed using the computer software (software) of Genetics Computer Group (University of Wisconsin, Center for Biotechnology, Madison, Wisconsin). Clone # 1441-10 was found to have an insert of approximately 1604 base pairs, and houses a long open reading structure (ORF) that codes for a protein of 142 amino acids. The full-length cDNA of this clone obtained from the rat tissue, called Neuritin, is described in Figure 1 (SEQ ID NO: 1). The amino acid sequence of Rat Neuritin is described in Figure 3 (SEQ ID NO 3). The cDNA of human Neuritin was cloned using the polymerase chain reaction (Pwo DNA polymerase and buffer, Boehringer Manheim) under standard conditions which were as follows: 5 minutes of denaturation at 94 ° C followed by 30 cycles of: 30 seconds at 94 ° C, 30 seconds at 56 ° C, and 30 seconds at 72 ° C using the following oligonucleotides: CTAGTCTAGAACCA GGGACT AG (SEQ ID NO 5) GGTATAGTCGACCCGTGCTCAGAA (SEQ ID NO.6) The template for this PCR reaction was Double-stranded cDNA which was generated from approximately 2 μg of human cortical mRNA (Clontech, Palo Alto, CA) using a Computer Marathon cDNA amplification (Clontech, Palo Alto, CA) following the manufacturer's recommendations. Amplified products of predicted size (approximately 435 base pairs) were subcloned into the cloning vector pCR-Script Amp SK (+) (Stratagene, La Jolla, CA) and both strands were sequenced using standard sequencing methods. The cDNA sequence of human Neuritin is shown in Figure 2 (SEQ ID NO 2). The predicted amino acid sequence of human Neuritin, based on the translation of the cDNA sequence, is described in Figure 4 (SEQ ID NO: 4). Analysis of the rat and human Neuritin protein reveals a putative, hydrophobic, amino-terminal signal peptide of about 24 amino acids. The C-terminal tail of 27 amino acids is enriched in hydrophobic residues and contains a consensual cleavage signal typically found in the GPI membrane anchored proteins (glucosyl-phosphotidyl-inositol). The protein bound to the membrane, mature 91 amino acids (approximately 12 kiloDaltones) and 6 cysteine residues. However, this amino acid sequence does not contain the general sequence or even the homology of the portion with any known protein, as evaluated by the sequential search and the public databases of DNA and protein (SWISS-PROT, PROSITE, GENBANK , and PIR), suggesting that it represents a new class of molecule family.

Example II: Preparation of Protelna Neuritin and Antibodies A rat Neuritin cDNA that codes for amino acids 30 to 113 of Neuritin ("Neuritin 30-113") was subcloned into the heat inducible bacterial expression vector pCFM1656 (ATCC accession number 69576) for the amplification and expression of Neuritin 30-113. The inclusion bodies containing Neuritin 30-113 were isolated by lysis of the bacteria in 3 ml of lysis buffer (50 mM Tris)., pH 8.0, 1 M EDTA, and 100 M NaCl containing 10 mg of lysozyme and 10 mg of sodium deoxycholate) per gram of bacteria. The bacteria used were treated with 400 μg of DNase I for 30 minutes at 1 hour, and then centrifuged at approximately 12,000 x g for 15 minutes at approximately 4 ° C. The inclusion bodies agglomerated by centrifugation were washed 2 to 4 times in 9 volumes of lysis buffer containing 0.5% NP-40. The purity of Neuritin 30-113 in the bodies of. Inclusion was assessed by SDS-PAGE. The purified inclusion bodies were solubilized (1:20) in urea 8 M, 50 mM Tris pH 8.0, 50 mM NaCl and 5 mM dithiothreitol (DTT) for 1 to 2 hours at room temperature (TA). The non-soluble material was removed by centrifugation at approximately 14 kg for 10 minutes at room temperature. The urea was dialyzed against 1 liter of the buffer described above, using the following time course and urea concentrations (all dialysis were performed at 4 ° C): 8 M urea at 6 M, 1 hour; 6 M to 4 M, all night; 4 M to 2 M, 1 hour; 2 M to 1 M, 1 hour; 1 M to 0.5 M, 1 hour; 0.5 M to 0.25 M, 1 hour, 0.25 M to 0 M, 1 hour. Neuritin 30-113 refolded was analyzed on non-reducing SDS-PAGE gels. To prepare antibodies for Neuritin, the peptide fragment of Neuritin: DCQEGAKDMWDKLRK (SEQ ID NO.7) which comprises an inner region of the mature Neuritin protein, was synthesized by standard methods and used for the immunization of rabbits (prepared by Berkeley Antibody Company, Berkeley, CA) resulting in the production of polyclonal antiserum called AS419. A second rabbit polyclonal antiserum was also prepared (designated AMG20) prepared against the fragment of Neuritin 30-113 expressed by bacteria, purified from the solubilized inclusion bodies, as described above (Cocalico Biologicals Inc., Reamstown, PA). Antisera that specifically reacted in Western blot analyzes of the recombinant Neuritin prepared in CHO cells were affinity purified using Sepharose beads containing the appropriate immobilized Neuritin peptide (Pierce Chemicals, Rockford, IL) followed by a column of A / G protein (Pierce Chemicals) to concentrate the antibody preparation. The rat and human recombinant neuronin spanning amino acids 1-115 was expressed in Chinese hamster ovary cells (CHO cells; ATCC accession number CRL-9096) by transfection of the cells with the pGREG plasmid containing either Rat or human neurotin. pGREG was prepared from the mammalian expression vector pDSRa2 (described in PCT patent application number WO 90/14363, published November 29, 1990). pGREG contains a sequence encoding a restriction enzyme site Xh ol, a thrombin cleavage site (see SEQ ID No. 8), an epitope of the herpes simplex virus recognized by the antibody in the 5 'to 3' direction. monoclonal of Novagen (Madison, Wisconsin), (see SEQ ID No. 9), an epitope of hexa-hi st idine for metal chelate chromatography, a stop codon, and a restriction enzyme site Sal.

LVPRGS (SEQ ID NO 8) QPELAPEDPEDVE (SEQ ID NO 9) The HSV / His tag was incorporated into pDSRa2 by stepped PCR using 4 overlapping oligonucleotides at the 3 'end of Neuritin and the 5' oligonucleotide described above (SEQ ID NO: 5). As the template for the PCR, pl441-10 was used. An initial oligonucleotide specific for the 3 'end of the rat Neuritin coding region incorporated the Xh ol and thrombin cleavage site. Three successive PCR reactions progressively incorporated the tagged sequence to the C-terminal end of rat Neuritin. The resulting product was subcloned into the Xba I and Sa l sites of pDSRa2. Additional tagged constructs were generated using PCR products containing the restriction sites Xba I and Xh or I. The human cDNA encoding amino acids 1 through 115 (and lacking the carboxyl-terminal GPI signal peptide) was used to express the secreted human Neuritin containing the thrombin cleavage site, the herpes simplex virus epitopes ( HSV) and hexa-Histidine (HIS) at its C-terminus. The human Neuritin cDNA lacking the GPI signal peptide (e.g., coding for amino acids 1-115) was generated by PCR using standard conditions and the following oligonucleotides.

CTAGTCTAGAACCATGGGACTTAAG (SEQ ID NO 10) GGTATACTCGAGCCCGTTGCCGCT (SEQ ID NO.11) The resulting product of 373 base pairs was subcloned into the Xba l and Xh ol sites of pGREG. The resulting vector is called pDSRahvl5Tag .1. The hexa-histidine tag allowed the easy purification of Neuritin on nickel-containing resin (Ni2 +) (Qiagen Inc., Chatsworth, CA). Media conditioned with CHO / Neuritin were prepared as follows. Revolving bottles containing CHO cells stably expressing the human labeled version of Neuritin 1-115 (designated hul5t36) were incubated at approximately 80 percent confluency (approximately 48 hours) in serum-free Dulbecco's Minimum Essential Medium (DMEM). The conditioned media were harvested by centrifugation of the cellular waste at approximately 2500 g; the supernatant was stored at -20 ° C. Neuritin was batch purified by incubating 1 ml of Ni2 + / NTA resin / 100 ml of conditioned medium, equilibrated with PBS (supplier of Ni27 NSAID: Qiagen Inc., Chatsworth, CA). The nonspecific proteins were removed by washing the resin with wash buffer (20 mM sodium phosphate, pH 6.0, and 500 M sodium chloride) containing increasing amounts of imidazole (20, 40, 80 and 100 mM) . Neuritin labeled with HSV-HIS specifically bound was eluted with 500 mM imidazole. The purified protein (more than 95 percent pure by SDS-PAGE with silver stain [BioRad Laboratories, Hercules, California] was concentrated to approximately one-tenth and diafiltered (Millipore Corp., [Ultra-free cut 15, cut] 5 K mw] Bedford, MA) in IX PBS The final concentration of the protein was estimated at 30-50 ng / ml using the Bio-Rad / Lowry protein assay with bovine serum albumin as a standard.

Example III: Neuritin Tissue Expression A. Northern Spotting Analysis To assess the expression pattern of Neuritin, a Northern blotting kit containing RNA from various rat tissues, including heart, brain, spleen, lung, liver, muscle, kidney, was purchased from Clontech (Palo Alto, Calif.). and testicles, and probed with 32P-labeled cRNA probes. The cRNA probes were generated from the cDNA of Rat Neuritin subcloned into pBluescript (Stratagene, La Jolla, CA) as follows. A fragment of approximately 430 base pairs of clone 1441-10 of Neuritin was obtained by digestion of the clone with Pvu II and Sma I. This fragment was subcloned into the pBluescript SK + plasmid (Stratagene, La Jolla, California) which was then named cpgl 5subclone # 2. To generate the antisense RNA probe, the plasmid was linearized with BamH1, after which transcription in vi tro was conducted using T7 polymerase (Promega, Madison, Wisconsin) and 3iP-UTP isolated essentially as described above using approximately 1 ml of guanidinium isothiocyanate lysis buffer. The amount of RNA on each spot was evaluated by periodic verification of the ethidium bromide staining of the RNA separated by size, and confirmed by hybridization of the stains with a randomly primed, labeled cDNA fragment of the ice-3-aldehyde glyphide. phosphate dehydrogenase (GAPDH). Northern analysis (RNA) as shown in Figure 5A identified a band of simple mRNA of approximately 1.6 kilobases expressed in the rat brain; a band of much lower intensity was observed in the lung tissue, and there was little or no hybridization to the mRNA of other tissues. To support the expression of Neuritin in various regions of the brain, adult rats were injected intraperitoneally with approximately 8 mg / kg of a stock solution of 10 mg / ml kainic acid in PBS. After about six hours, the rats were sacrificed and the brain tissue was dissected. RNA was isolated from various regions of the brain using approximately 1 ml of guanidinium isothiocyanate lysis buffer (Chomczynski et al., Ana l. Bi och em. , 162: 156 [1987]) per 100 mg of powdered tissue. After lysis, the solution was passed over silica gel membrane columns (rotating columns RNeasy, Qiagen, Chatsworth, California). The RNA was fractionated by size by separation on agarose gels with 0.8-1% formaldehyde and capillary staining on nylon membranes (Hybond-N, Amersham, Arlington Heights, IL). The Northern blots were probed with "P" labeled cDNA probes as described above As can be seen in Figure 5B, the dentate gyrus region of the brain had the highest level of Neuritin expression.

B. Hybridization In Si t u e Immunohis toquímica In si t u hybridization and immunohistochemistry were performed on rat embryonic tissue and adult rat brain tissue sections, which were fixed with paraformaldehyde and embedded in paraffin as follows. Embryos from pregnant rats were isolated and fixed overnight in 4% fresh paraformaldehyde in PBS (4% PFA / PBS) at 4 ° C before dehydration and paraffin embedding. The brains of adult rats were prepared by transcardiac perfusion of the anesthetized animals, with 4% paraformaldehyde in PBS. The dissected brains were then fixed overnight at 4 ° C in 4% paraformaldehyde in PBS, dehydrated, and embedded in paraffin. Hybridization in si tu on these tissue sections was performed according to established methods (Simonet et al, J. Biol. Ch em., 11: 8221-8229 [1993]) using a rat Neuritin cRNA probe prepared as described above for Northern blots, except that 32S-UTP was used instead of 32P-UTP. The hybridized slides were exposed to Kodak photographic emulsion and developed after 3 to 6 weeks, after which time the sections were counter stained with hematoxylin, and the silver beads were visualized using dark field optics. The immunohistochemical localization of Neuritin was conducted using various dilutions of affinity purified antiserum (AS-419 or AMG20, described above), specific for human recombinant Neuritin prepared in mammalian cells, on tissue sections fixed with deparaffinized PFA. Neuritin antibody, bound, was detected with goat anti-rabbit immunoglobulin, biotinylated and avidin labeled with horseradish peroxidase, using the Vectastain Elite ABC staining equipment (Vector Labs, Burlingame, CA) according to the manufacturer's instructions . The in si t u hybridization analysis showed that Neuritin mRNA is present at the boundary between the neuroepi telium and the differentiation zone of the developing rat brain, as early as on embryonic day 14 (E14). The message was also located in the developing neuronal structures in the periphery, including the dorsal root ganglia and the trigeminal ganglia. The expression seemed to increase throughout the development, and it seems to become more concentrated within the zone of differentiation as the individual structures within the central nervous system become more defined. Neuritin mRNA was detected in most adult brain structures. The majority of abundant signals were found in layers II-IV of the cortex, in the hippocampal formation, in the thalamus, in the habenula and in the brainstem. In the hippocampus, the expression was concentrated in the neurons of the pyramidal and granular cell layer, with abundant levels found in neurons of the subicular and spinning region of the dentate region. In the cerebellum, low levels of Neuritin message were located in the granular cell layer with dotted dotted scattering of Purkinj e cells.

Immunohistochemical staining of brain tissues using Neuritin AS419 or AMG20 antibodies, described above, shows that Neuritin is concentrated on the bodies of neuronal cells and disperses non-uniformly along neuritic projections in unmyelinated regions of the brain . Uneven staining results in a granular appearance of the immunoreactive regions, and is particularly evident along the projections of neurons in the hippocampal subicular complex. The concentration of Neuritin along the neurites is also documented in the staining of the dendritic trees of the positive purkinje cells. The spinning region of the DG contains strongly dispersed immunoreactive cells that correlate with the pattern observed by in si t u hybridization of Neuritin mRNA. Irregular staining of purkinje neurons also correlates with the location of the dotted message.

Example IV: Neuritin Biochemistry and Regulation of Expression The amino acid sequence of the carboxyl terminus of Neuritin suggested the possibility that Neuritin is anchored to the membrane. To evaluate this possibility, approximately 1 x 10c CHO cells transfected with either an empty plasmid (termed "progenitor" and not containing the Neuritin gene) or with a plasmid containing the gene encoding human Neuritin, were treated either with approximately 0.4 U / ml of PI-PLC (phosphatidyl-inositol-phospholipase C; Calbiochem, La Jolla, California) prepared in 0.5 ml of release buffer (25 mM Tris-HCl, pH 7.5, 1 mM EDTA, glucose 10 mM, 250 mM sucrose), or with the release buffer alone (without PI-PLC) following the published methods (Kodukula et al., J. Cel l Bi., 120: 657 [1993]). After incubation, the cells were centrifuged to precipitate cell debris. The supernatants were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gels were stained on nitrocellulose paper and probed with affinity purified antiserum, specific for Neuritin. The results are shown in Figure 8A. As can be seen, all the detectable Neuritin was found in the supernatants of the CHO cells expressing Neuritin that had been treated with PI-PLC, suggesting that Neuritin is rather anchored to GPI. The analysis of this endogenous Neuritin anchored to GPI was carried out by means of the extraction of the tissue with the Triton X-114 detergent (Calbiochem, San Diego, California) according to the method described by Borchelt et al.

(Glycobi ol. 3: 319 [1993]) to obtain native Neuritin. Approximately suspended 100 mg of powdered tissue in 0.5 ml of lx TNE buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA) with protease inhibitors (0.5 mM benzamidine, 1 mM PMSF, and 1 μg / ml each of pepstatin, leupeptin, aprotinin) and mixed at 4 ° C for approximately 0.25 volumes of Triton X-114 pre-equilibrated with IX TNE. Proteins soluble in Triton X-114 were extracted from the solution by incubating the mixture at 30 ° C for approximately 15 minutes, followed by centrifugation at approximately 3000 g for 5 minutes at room temperature to precipitate the micelles of detergent containing protein, lipophilic, large. This extraction was repeated and the soluble fractions containing detergent were combined. To analyze the proteins extracted by Western blotting, aliquots of 20-40 μl of the detergent fractions were precipitated by incubating each aliquot with 10 volumes of methanol (10 minutes at 4 ° C followed by centrifugation at 14,000 g for 15 minutes at 4 ° C). The pellet was resuspended in approximately 40 μl of SDS-PAGE sample buffer (containing be ta-mercaptoethanol), and fractionated by size on a 16 percent SDS-PAGE gel (Novex, San Diego, California). After electrophoresis, the proteins were transferred onto nitrocellulose paper using standard Western staining procedures. Neuritin was detected using affinity-purified antisera, specific for Neuritin (AS419 or AMG20, described above) as a first antibody, and goat anti-rabbit antisera, conjugated to horseradish peroxidase as a second antibody (diluted approximately 1: 104, obtained from Southern Biotechnology Associates, Inc., Birmingham, Alabama). Antibodies were detected using the enhanced, peroxidase-sensitive chemiluminescence ("ECL", Amersham, Arlington Heights, IL). The results are shown in Figure 8B. As it is apparent, the regions of the cortex and hippocampus of the brain expressed the highest levels of Neuritin. Neuritin from recombinant CHO cells, prepared as described above using PI-PLC is shown in band 1 as a comparison. The difference in the migration of Neuritin in the bands on the Western blot is presumably due to the altered mobility of Neuritin to which some lipid is coupled. To investigate the regulation of Neuritin mRNA expression, recombinant human BDNF, NT-3- NGF, or FGF (approximately 10 ng / ml), KCl (approximately 50 mM), or calcium channel blockers or MAP NMDA (approximately 10 μM) were added to hippocampal cultures or rat cortices 7DIV E18, prepared as described below in Example V. The RNA was isolated from each culture after approximately six hours of incubation by the RNeasy method (Qiagen, Chatsworth, California). Approximately 5 μg of this RNA was loaded on a gel, separated by electrophoresis, and then stained by Northern. The spot was probed with a Neuritin cRNA probe spanning the coding region (described above). The results are shown in Figure 6A. As can be seen, treatment with NMDA and AMPA resulted in an approximately 5-fold increase in Neuritin mRNA levels, and a similar magnitude of induction was observed with depolarizing concentrations of KCl. To evaluate the effects of BDNF on the expression levels of Neuritin i n vi vo, BDNF (1-2 μl of 10 mg / ml) or saline (control) were injected intraventricularly into 4-day postnatal rat pups. Approximately six hours after the administration of BDNF or saline, the pups were sacrificed and the total RNA was isolated from the cortex or hippocampal brain tissue. As can be seen in Figure 6B, BDNF induced the message of Neuritin in vi, mainly in the hippocampus, and to a small degree in the cortex.

Example V: Neuritine Bioactivity Assays Cultures of primary hippocampal and primary cortical embryonic rat neurons were prepared by dissociating the dissected brain regions from 18-day-old rat embryos. Dissociation of the purification of the embryonic neurons was conducted using a papain-based tissue dissociation kit (Worthington biochemical, Corp., Freehold, NJ). The dissected tissue from 10-30 embryos was resuspended in 2.5 ml of Earles balanced saline solution (EBSS) containing: 50 units of papain, 1 mM L-cysteine, 0.5 mM EDTA and 500 units of DNase I). The tissue was dissociated for 10 to 15 minutes with gentle agitation. The dissociated cells were centrifuged at 300 g for 5 minutes, resuspended in 2.7 ml of EBSS, 0.3 ml of ovomucoid inhibitor solution (10 mg / ml of ovomucoid protease inhibitor and 10 mg / ml of bovine serum albumin) and 250 units of DNase I The suspension was spread over 5 ml of ovomucoid inhibitor solution and centrifuged at 70 g for 6 minutes. The concentrated cells were resuspended in 10 ml of neurobasal medium containing B27 (Gibco / BRL, Grand Island, NY) and passed through a 40 μm nylon mesh cell (Becton Dickinson, Lincoln Park, NJ). For RNA analysis the dissociated neurons were plated on 6-well Falcon tissue culture plates (Becton Dickinson, Lincoln Park, NJ) pre-coated with poly-L-ornithine (obtained from Sigma, Saint Louis, MO , and used at a concentration of approximately 0.1 mg / ml in 150 mM sodium borate, pH 8.4) and laminin (obtained from Gibco / BRL, Grand Island, NY, and used at a concentration of approximately 1 μg / ml in PBS) . The plaque placement of the neurons was at a density of approximately 2x10 ^ per cm "for hippocampal neurons and approximately 3x05 per cm 'for cortical neurons.The cells were developed in Neurobasal media (Gibco / BRL, Grand Island, NY ) supplemented with IX of B-27 (Gibco / BRL, Grand Island, NY) and 50 mg / ml of gentamicin sulfate (Gibco / BRL, Grand Island, NY) The total cell content of each culture after seven days culture was less than five percent, as assessed by counting cells positive for the glial fibrillary acidic protein (GFAP), which were identified by indirect immunofluorescence staining using the antibody specific for this specific marker of the gual cells. Cells were treated as described, after 7 to 8 days of culture.The neurite development assay was conducted using hippocampal and cortical neurons prepared as described above. The cells were placed in a plate on a 35 mm plate coated with poly-lysine (20 μg / ml in PBS) at a density of approximately x 10J cells per cm "in the presence or absence of Neuritin purified with Ni "* (prepared as described above) The neurite development was evaluated after four days of culture by staining the live cultures with the non-specific lipophilic dye, Dil (10 μM, visualized with 565 filter nm), for approximately 30 minutes at 37 ° C, followed by 3 washes in Neurobasal medium supplemented with B-27 (see above) before the analysis To examine the biological function of Neuritin, a histidine-labeled version of Neuritin that lacks the 27 carboxyl-terminal amino acids, was produced in Chinese hamster ovary (CHO) cells and purified from serum-free conditioned medium by Ni2 + affinity chromatography at a homogeneity greater than ninety percent, as determined by staining with silver SDS-polyacrylamide gels (see above). The hippocampal and cortical neurons of rat E18 embryos were plated on coated discs. coated with poly-lysine in the presence of approximately 150 ng / ml of this recombinant Neuritin. The same volume was added to the controls using an equivalent i '"affinity fraction, derived from the conditioned media from the CHO cells transfected with the empty expression vector." After four days in culture, the neurons plated in Neuritin presence showed neuritogénes is extensive over the control cultures, as shown in Figure 9. The cells treated with Neuritin had more, and larger highly branched neuritic trees, and an increased number of neurites extending from the soma into Comparison to control cells Cells that were not specifically stained with the lipophilic fluorescent dye Dil, revealed a strong difference in the organization of the soma and the neurotic lamellapodia (Figure 9) .The untreated control cells had flat and wide cell bodies, lamelapodia seemingly unfocused along the length or towards the end of many neurites, while cells treated with Neuritin had well-differentiated cell bodies with thin, well-defined extensions. The cellular neurotogenic acti vity was observed with the purified bacterial neuritin.

Neuritin cDNA deposit The cDNA encoding full length human Neuritin has been deposited with the ATCC (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD, USA) on August 9, 1996 as accession number 98134.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: Amgen Inc. (Ü) TITLE OF THE INVENTION: NEW NEUROGEN (iii) SEQUENCE NUMBER: 11 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: AMGEN INC. (B) STREET: 1840 DEHAVILLAND DRIVE (C) CITY: THOUSAND OAKS (D) STATE: CA (E) COUNTRY: USA (F) POSTAL CODE: 91320-1789 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Diskette (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0. Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: 08 / 694,579 (B) DATE OF SUBMISSION: 09 AUGUST 1996 (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: OLESKI, NANCY A. (B) REGISTRATION NUMBER: 34,688 (C) REFERENCE NUMBER / CASE: A-413 (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1604 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: ACTCTCTCGC CTCTTTCTG TCTCTTCCTC GCTCCCTCTC TTTCTCTCCT CCCTCTGCCT 60 TCCCAGTGCA TAAAGTCTC GTCGCTCCCG GAAC TGTTG GCAATGCCTA TTTTTCAGCT 120 TTCCCCCGCG TTCTCTAAAC TAACTATTTA AAGGTCTGCG GTCGCAAATG GTTTGACTAA 180 ACGTAGGATG GGACTTAAGT TGAACGGCAG ATATATTTCA CTGATCCTCG CGGTGCAAAT 240GTGCAGGCCG TGAGAGCAGC AGGCAAGTGC GATGCAGTCT TTAAGGGCTT 300 TTCAGACTGT TTGCTCAAGC TGGGTGACAG CATGGCCAAC TACCCGCAGG GCCTGGACGA 360 CAAGACGAAC ATCAAGACCG TGTGCACATA CTGGGAGGAT TTCCACAGCT GCACGGTCAC 420 AGCTCTTACG GATTGCCAGG AAGGGGCGAA AGATATGTGG GATAAACTGA GAAAAGAATC 480 GAAAAACCTC AATATCCAAG GCAGCTTATT CGAACTCTGC GGCAGCGGCA ACGGGGCGGC 540 GGGGTTCTG CTCCGGCGC TTTCCGTGCT CCTGGTGTCT CTCTCGGCAG CTTTAGCGAC 600 C GGCTTTCC TTCTGACTTC TGAGCACGGG GCCGGGTCCC CCCTCCGCTC ACCCACCCAC 660 ACTCACTCCA TGCTCCCGGA AATCGAGAGG AAGAGCCATT CGTTCTCTAA GGACGTTGTG 720 ATTCTCTGTG ATATTGAAAA CACTCATATG GGATTGTGGG AAATCCTGTT TCTCTCTTTT 780 TTTTT TTTA ATTTTTTTTTT ATTTTGGTTG AGTCCTTGTG TTTTAGTTGC CAAATGTTAC 840 CGATCAGTGA GCAAAGCAAG CACAGCCAAA ATCGGACCTC ACCTTAAGTC CGTCTTCACA 900 CAAAAATAAG AAAACGGCAA ACTCACCCCC ATTTTTAATT TTGTTTTTAA TTTTACTTAC 960 TTATTTATTT ATTTATTTTT TGGCAAAAGA ATCTCAGGAA TGGCCCTGGG CCACCTACTA 1C20 TATTAATCAT GTTGATAACA TGAAAAATGA TGGGCTCCTC CTAATGAGAA AGCGAGGAGA 1080 GGAGAAGGCC AGGGGAATGA GCTCAAGAGT GATGCCCACG TGGGAATAAT CGCTCACGTC 1140 TTTCTTCCAC AGTACCTTGT TTTGATCATT TCCACAGCAC ATTTCTCCTC CAGAAACGCG 1200 AAAAACACAA GCGTGTGGGT TCTGCATTTT TAAGGATAAG AGAGAGAAAG AGGTTGGGTA 1260 TAGTAGGACA GGTTGTCAGA AGAGATGCTG CTATGGTCAC GAGGGGCCCCG TTTCACCTGC 1320 TATTGTCGTC GCCTCC TCA GTTCCACTGC CTTTATGTCC CCTCCTCTCT CTTGTTTTAG_1380_CTGTTACACA TACAGTAATA CCTGAATATC CAACGG ATA GTTCACAAGG GGGTAATCAA 1 40 TGTTAAAtC AAAATAGAAT TTAAAAAAAA AAGATTTTGA CATAAAAGAG CCTTGATTTT 1500 AAAAAAAAAG AGAGAGATGT AATTTAAAAA GTTTATTATA AAT AAATTC AGCAAAAATT 1560 7GCTACAAAG TA AGAGAAG TATAAAATAA AAGTTATTGT TTGA 1604 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 435 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (II) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: ATGGGACTTA AGTTGAACGG CAGATATATT TCACTGATCC TCGCGGTGCA AATAGCGTAT 60 CTGGTGCAGG CCGTGAGAGC AGCGGGCAAG TGCGATGCGG TCTTCAAGGG CTTTTCGGAC 120 TGTTTGCTCA AGCTGGGCGA CAGCATGGCC AACTACCCGC AGGGCCTGGA CGACAAGACG 180 AACATCAAGA CCGTGTGCAC ATACTGGGAG GATTTCCACA GCTGCACGGT CACAGCCCTT 240 ACGGATTGCC AGGAAGGGGC GAAAGATATG TGGGATAAAC TGAGAAAAGA ATCCAAAAAC 300 CTCAACATCC AAGGCAGCTT ATTCGAACTC TGCGGCAGCG GCAACGGGGC GGCGGGGTCC 360 CTGCTCCCGG CGTTCCCGGT GCTCCTGGTG TCTCTCTCGG CAGCTTTAGC GACCTGGCTT 420 TCCTTCTGAG CACGG 435 (2) INFORMATION FOR SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 142 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 3: Mer Wing Asn Tyr Pro Gln Gly Leu Asp Asp Lys Thr Asn lie Lys Th < - 5 ° 55 60 Val Cys Thr Tyr Trp Glu Asp Phe His Ser Cys Thr Val Thr Ala Leu 65 70 75 8Q Thr Asp Cys Gln Glu Gly Wing Lys Asp Mee Trp Asp Lys Leu Arg Lys 85 90 95 Glu Ser Lys Asn Leu Asn lie Gln Gly Ser Leu Phe Glu Leu Cys Gly 100 105 not Ser Gly Asn Gly Ala Wing Gly Ser Leu Leu Pro Wing Leu Ser Val Leu 115 120 125 Leu Val Ser Leu Ser Ala Ala Leu Ala Thr Trp Leu Ser Phe 130 135 1 0 (2) INFORMATION FOR SEQ ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 142 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4: Mee Gly Leu Lys Leu Asn Gly Arg Tyr He Ser Leu lie Leu Ala Val 1 5 10 15 Gln lie Wing Tyr Leu Val Gln Wing Val Arg Wing Wing Gly Lys Cys Asp 20 25 30 Wing Val Phe Lys Gly Phe Ser Asp Cys Leu Leu Lys Leu Gly Asp Ser 35 40 45 Met Wing Asn Tyr Pro Gln Gly Leu Asp Asp Lys Thr Asn He Lys Thr 50 55 60 Val Cys Thr Tyr Trp Glu Asp Phe His Ser Cys Thr Val Thr Ala Leu 65 70 75 80 Thr Asp Cys Gln Glu Giy Wing Lys Asp Met Trp Aep Lys Leu Arg Lys 85 90 95 Giu Ser Lys Asn Leu Asn He G n Gly Ser Leu Phe Glu Leu Cys Gly 100 105 110 Ser Gly Asr. Gly Ala Ala Gly Ser Leu Leu Pro Ala Phe Pro Val Leu 115 120 125 Leu Val Ser Leu Ser Ala Ala Leu Ala Thr Trp Leu Ser Phe 130 135 140 (2) INFORMATION FOR SEQ ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / dése = "SYNTHETIC DNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 5: CTAGTCTAGA ACCATGGGAC TTAAG 25 (2) INFORMATION FOR SEQ ID NO. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "SYNTHETIC DNA" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 6: GGTATAGTCG ACCCGTGCTC AGAA 24 (7) INFORMATION FOR SEQ ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 7: sp Cys Gln Glu Gly Ala Lys Asp Met Trp Asp Lys Leu Arg Lys 5 10 15 (2) INFORMATION FOR SEQ ID NO. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 8: Leu Val Pro Arg Gly Ser 1 5 (2) INFORMATION FOR SEQ ID NO. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 9: Gln Pro Glu Leu Wing Pro Glu Asp Pro Glu Asp Val Glu 1 5 10 (2) INFORMATION FOR SEQ ID NO. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "SYNTHETIC DNA" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 10: CTAGTCTAGA ACCATGGGAC TTAAG 25 (2) INFORMATION FOR SEQ ID NO. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "SYNTHETIC DNA" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 11: GGTATACTCG AGCCCGTTGC CGCT 24 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (23)

1. A nucleic acid molecule, characterized in that it encodes a polypeptide selected from the group consisting of: (a) the nucleic acid molecule of SEQ ID NO. 1; (b) the nucleic acid molecule of SEQ ID NO. 2; (c) a nucleic acid molecule encoding the polypeptide of SEQ ID NO. 3; (d) a nucleic acid molecule encoding the polypeptide of SEQ ID NO. 4; (e) a nucleic acid molecule encoding a polypeptide that is at least 70 percent identical to the polypeptide of SEQ ID NO. 3 or of SEQ ID NO. 4; and (f) a nucleic acid molecule that is the complement of any of (a) - (e) above.

2. The nucleic acid molecule, characterized in that it is SEQ ID NO. 1.

3. The nucleic acid molecule, characterized in that it is SEQ ID NO. 2.

4. A nucleic acid molecule, characterized in that it encodes the polypeptide of SEQ ID NO. 3.

5. A nucleic acid molecule, characterized in that it encodes the polypeptide of SEQ ID NO. Four.

6. A vector, characterized in that it comprises the nucleic acid molecule according to claim 1.

7. A vector, characterized in that it comprises the nucleic acid molecule according to claim 2.

8. A vector, characterized in that it comprises the nucleic acid molecule according to claim 3.

9. A vector, characterized in that it comprises the nucleic acid molecule according to claim 4.

10. A vector, characterized in that it comprises the nucleic acid molecule according to claim 5.

11. A host cell, characterized in that it comprises the vector according to claim 6.

12. A host cell, characterized in that it comprises the vector according to claim 7.

13. A host cell, characterized in that it comprises the vector in accordance with 15 rei indication 8.

14. A host cell, characterized in that it comprises the vector according to claim 9.

A host cell, characterized in that it comprises the vector according to claim 10.

16. A process for the production of a Neuritin polypeptide, characterized in that it comprises the steps of: (a) expressing a polypeptide encoded by the nucleic acid according to claim 1 in an appropriate host; and (b) isolation of the polypeptide.

17. The process according to claim 16, characterized in that the polypeptide is SEQ ID NO. 3 or SEQ ID NO. Four.

18. A Neuritin polypeptide, characterized in that it is selected from the group consisting of: (a) the polypeptide of SEQ ID NO. 3; (b) the polypeptide of SEQ ID NO. 4; and (c) a polypeptide that is at least 70 percent identical to the polypeptide of (a) or (b).

19. A Neuritin polypeptide, characterized in that it is the polypeptide of SEQ ID NO. 4 or a biologically active fragment thereof.

20. The Neuritin polypeptide according to claim 19, characterized in that it does not possess an amino-terminal methionine.

21. The polypeptide according to claim 20, characterized in that it is selected from the group consisting of: amino acids 25-115; amino acids 1-115; and amino acids 1-113.

22. An antibody or fragment thereof, characterized in that it binds specifically to human Neuritin.

23. The antibody according to claim 22, characterized in that it is a monoclonal antibody.