EP1685151A2 - Neural regeneration peptides and methods for their use in treatment of brain damage - Google Patents

Abstract

The invention discloses a family of peptides termed NRP compounds or NRPs that can promote neuronal migration, neurite outgrowth, neuronal proliferation, neural differentiation and/or neuronal survival, and provides compositions and methods for the use of NRPs in the treatment of brain injury and neurodegenerative disease. NRP compounds can induce neurons and neuroblasts to proliferate and migrate into areas of damage caused by acute brain injury or chronic neurodegenerative disease, such as exposure to toxins, stroke, trauma, nervous system infections, demyelinating diseases, dementias, and metabolic disorders. NRP compounds may be administered directly to a subject or to a subject's cells by a variety of means including orally, intraperitoneally, intravascularly, and directly into the nervous system of a patient. NRP compounds can be formulated into pharmaceutically acceptable dose forms for therapeutic use. Methods for detecting neural regeneration, neural proliferation, neural differentiation, neurite outgrowth and neural survival can be used to develop other neurally active agents.

Description

NEURAL REGENERATION PEPTIDES AND METHODS FOR THEIR USE IN TREATMENT OF BRAIN DAMAGE

Related Applications This application claims priority to U.S. Provisional Application Serial No:

60/516,018, filed October 31, 2003, titled "Neural Regeneration Peptides and Methods for Their Use," Frank Sieg and Thorsten Gorba, inventors (Attorney Docket No: NRNZ 01023 US3 DBB), to U.S. Provisional Application Serial No: 60/585,041, filed July 2, 2004, titled "Neural Regeneration Peptides: A New Class of Chemoattractive and Neuronal Survival Promoting Peptides," Thorsten Gorba and Frank Sieg, inventors (Attorney Docket No: NRNZ 01023 US4 DBB), and U.S. Provisional Application Serial No: 60/616271, titled: "Neural Regeneration Peptides and Methods for Their Use in Treatment of Brain Damage," Frank Sieg, Paul Edmond Hughes and Thorsten Gorba inventors, filed October 5, 2004 (Attorney Docket No: NRNZ 01023 US5 DBB). All of the above applications are incorporated into this application fully by reference.

Sequence Listing: This application contains a sequence listing presented as (1) a printed copy of the Sequence Listing and (2) a diskette containing the Sequence Listing in computer readable form. The Sequence Listing is incoφorated into this application fully by reference.

Field of the Invention

Tins invention is directed to compositions and methods for the use of oligonucleotides and peptides that promote neuronal migration, proliferation, survival, differentiation, and/or neurite outgrowth. More specifically, this invention is directed to the use of such peptides in the treatment of brain injury and neurodegenerative disease. This invention also includes new methods for detecting neural cell growth, migration, neurite outgrowth, survival and/or differentiation.

BACKGROUND Related Art Mild to severe traumatic brain injury (TBI), and focal or global ischemia can result in significant neuronal cell loss and loss of brain function within a short time period after the insult. There are no treatments currently available to prevent cell death that occurs in the brain as a consequence of head injury or damage caused by disease. To date, there is also no treatment available to restore neuronal function. Treatments available at present for chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and Multiple Sclerosis only target symptoms. No drugs are currently available to intervene in the disease process or prevent cell death. It is well known that cortical-subcortical non-thalamic lesions can lead to apoptosis within thalamic areas 3-7 days after an insult. Retrograde thalamic degeneration is accompanied by activation of astroglia and microglia in the thalamus (Hermann et al., 2000). Non-invasive techniques like MRI reveal smaller thalamic volumes and increased ventricle-to brain ratio values within TBI patients suffering from non-thalamic structural lesions (Anderson et al., 1996). These findings indicate the high vulnerability of thalamocortical excitatory projection neurons for retrograde-triggered neuronal cell death and therefore indicate the need for a rescue strategy of injured or insulted thalamic neurons. Functioning of the inhibitory neuronal circuits within the thalamus is crucial for intrathalamic down regulation of neuronal activity within the thalamus as well as within the striatal system. It has been shown that animals with striatal lesions similar to those that occur in Huntington's disease show an improvement in behavioural outcome when GABA-releasing polymer matrices are implanted into the thalamus (Rozas et al., 1996). On a cellular level within the striatum it has been shown that calbindin immunoreactive ("calbindin-ir") inhibitory neurons can be rescued by administering activin A (Hughes et al., 1999). Until now, only transplantation involving fetal striatal implants lead to an improvement or restoration of motor functions in Huntington's disease animal models (Nakao and Itakura, 2000). Restoring thalamic and striatal GABA-ergic systems that are impaired during Huntington's disease, can improve behavioural outcome (Beal et al., 1986). A feature of the developing nervous system is the wide-ranging migration of precursor cells to their correct three-dimensional spatial position. These migrations promote differentiation of an array of phenotypes and the arrangement of immature neurons into the vertebrate brain. To achieve the correct wiring of approximately 100 billion neurons, construction of a cellular organisation like the formation of laminar structures in higher cortical regions is necessary (see Hatten and Heintz, 1999 for a review). A cellular correlate for the direction of movement of a migrating neuron may be the frequency and amplitude of transient Ca²⁺ changes within a single migrating cell (Gomez and Spitzer, 1999) although the triggering of initiation and/or commitment of neuronal cell migration by membrane-bound or diffusible molecules remains elusive. Many of the cues that are involved in neurite outgrowth and neuronal migration, however, have been identified. Plasma membrane molecules belonging to the integrin receptor family interact with extracellular matrix ligands, like laminin, to initiate neuronal adhesion to the substratum (Liang and Crutcher, 1992; De Curtis and Reichardt, 1993). The control of integrin expression affects a wide range of developmental and cellular processes, including the regulation of gene expression, cell adhesion, neurite outgrowth and cell migration. Other ligands which promote cell migration are cell adhesion molecules (i.e. N- CAM; cadherins; TAG-1), the laminin-like molecule netrin-1, the neuron-glial adhesion ligand astrotactin and growth or neurotrophic factors such as EGF, TGF-α, platelet activating factor and BDNF (Dodd et al., 1988; Yamamoto et al, 1990; Ishii et al., 1992; Ferri and Levitt, 1995; Ganzler and Redies, 1995). Recently, collapsin-1 (semaphorin3A) was discovered. Collapsin-1 has chemorepulsive activities in axonal guidance and migration patterns for primary sensory neurones (Pasterkamp et al, 2000). In contrast, collapsin-1 acts as a chemoattractant for guiding cortical apical dendrites in neocortical areas (Polleux et al., 2000). Similar chemorepulsive as well as chemoattractive effects on axonal guidance are displayed by slit-1, a diffusible protein (Brose et al., 2000). Currently, the cascade leading to the initiation of neuronal movement, namely adhesion of the neuron followed by initiation of migration, the process of migration over long distances, including turns and the migration stop signal remains to be elucidated. Midbrain lesions with simultaneously administered TGF-α lead to a massive proliferation of multipotential stem cells originating in the subventricular zone ("SVZ") and subsequent migration of these progenitor cells into the striatum (Fallon et al., 2000). It may be desirable, however, to activate neuronal proliferation and migration of neurons that are in close vicinity to the site of a lesion in order to prevent long-distance migration of neuronal precursors originating from the SVZ. There is only one report featuring the chemokine stromal-derived factor (SDF-1) as a neuronal migration chemoattractant. The embryonic expression pattern of SDF-1 attracts cerebellar granule cells to migrate from the external germinal layer to the internal granular layer (Zhu et al., 2002). Nevertheless, this chemokine has no influence on postnatal tissue. There are no known migration-inducing factors that have direct chemoattractive effects on the migration behaviour of neuroblasts or neurons in adults after brain trauma or neurodegenerative disease.

SUMMARY OF THE INVENTION It is therefore an object of embodiments of the present invention to provide new approaches to the treatment of brain injuries and diseases. Such embodiments include peptides that can induce one or more of neural migration, neural outgrowth, neural proliferation, neural differentiation and/or neural survival. These peptides are herein termed "neural regeneration peptides" or "NRPs." Other embodiments include administration of one or more NRPs following brain injury or during chronic neurodegenerative disease. The term "NRP" or "NRP compound" includes NRPs, NRP homologs, NRP paralogs, NRP orthologs and/or NRP analogs. An NRP can either be administered alone or in conjunction with one or more other NRPs or with other types of agents to promote neural outgrowth, neural migration, neural survival, neural differentiation and/or neural proliferation. NRPs and related peptides generally have certain amino acid sequences (also termed

"domains") present, which confer desirable biological properties on the molecule. Some embodiments of NRP peptides with certain domains highlighted are shown in

Table 1 below. Table 1 Neural Regeneration Peptides*

NRP-l: YD P EAA S - AP G S GN P - - - - - - C H

NRP-2KG: KD P E A R R - A P G S H P - - - - - - C - - L AA - S C S AA G NRP-3SF: S D S F K S Q - A R G Q V P P F L G GV G C P F

NRP-4GG: G T P G RA E - A G G Q V S P - - - - - - C - - L AA - S C S Q A Y G NRP-5RP2:R E - - G R R D A P G RA - - G G G G - - - - - - AA R S V S P S P NRP-7S : S E P E AR R - AP G R K - - - - G GVV CA S LAAD

NRP-8SG: S E V D A R R - A K K S H - - - - - - - C - I L S - D T S H P R G

NRP-9SD: S E P E A R RA Q G G Q I P S E RV S D In some embodiments, NRPs generally comprise a chain length of between about 8 to about 25 amino acids and having molecular weights between about 0.8 and about 2.7 kDa. Additionally, in other embodiments, an NRP can have an isoelectric point between about 6.5 and about 10.0, and having at least one biological property promoting an outcome selected from neuronal survival, neurite outgrowth, neuronal proliferation, neuronal differentiation and neuronal migration. Additionally, an NRP may have one or more domains, as indicated in bold in Table 1 above. In some embodiments, an NRP may have a [A]PG[R,S] domain in combination with a PE-domain (e.g., NRP-l and NRP-2) or alternatively, without a PE- domain (e.g., NRP-5, NRP-7). The presence of a [A]PG[R,S] domain is desirable for NRP biological activity. Additionally a C-terminal GG domain can confer desirable neuroprotective properties on a NRP. Thus, in alternative embodiments, NRPs can have a first domain selected from the group consisting of a [A]PG[R,S] domain, an [A,G]RR domain and an ARG domain have desirable biological activity. In other embodiments, desirably, an NRP can have, in addition to a first domain as described above, a second domain different from the first domain. A second domain can be a PE domain an [A,G]RR domain or a C- terminal GG domain. In certain further embodiments, NRP s can have a third domain of those described above. Thus, in certain embodiments, an NRP may have a [A]PG[R,S] domain alone, other NRP can have an ARG domain alone, still other NRPs can have an [G,A]RR domain alone. Still other NRPs can have a [A]PG[R,S] domain and a PE domain, and still other NRPs can have a [A]PG[R,S] domain and a [G,A]RR domain. Still other NRPs can have a [A]PG[R,S] domain, an [A,G]RR domain and a PE domain. Genes of NRP family members contain at least one of a CAAT-Box and a TATA- Box, or both CAAT-Box and TATA-Boxes together in promoter regions. Oligonucleotides derived from NRP family members can be used to increase expression of NRP peptides in regions where such production is desired. In another aspect, embodiments of this invention provide methods of treatment for damaged areas of the brain as a consequence of head injury or chronic neurodegenerative disease by administering one or more NRPs, NRP analogs (including peptides with structural similarities) and/or NRP prodrugs (including pro-NRP peptides) to promote neuronal or neuroblast migration, proliferation, survival and/or neurite outgrowth. This method of treatment may be particularly useful but in no way limited to, patients suffering from mild to severe traumatic brain injury (TBI) that involves neocortical damage as well as injuries to subcortical areas. In one embodiment, NRP-2 (SEQ ID NO: 5) is encoded by a nucleic acid sequence localised on human chromosome 13 within the genomic clone bA87Gl (Sanger Sequencing Centre) on the reverse strand between base pairs 77232-76768. This peptide has functions similar to those of rat NRP-l and can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration and neurite outgrowth. In another embodiment, NRP-3 (SEQ ID NO: 7) is encoded by a nucleic acid sequence localized on the reverse strand of chromosome 3 in the human genome, between base pairs 34764-33003 according to Double Twist annotation. This NRP also can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. In still another embodiment, NRP-4 (SEQ ID NO: 9) is encoded by a nucleic acid sequence located between base pairs 21970003-21972239 on the forward strand of human chromosome 15, according to the NCBI human genome annotation project. Peptides translated from that nucleic acid sequence also belong to the human family of NRPs. Peptides encoded by this sequence can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. A still further embodiment, NRP-5 (SEQ ID NO: 11), is encoded by a nucleic acid sequence localized on the reverse strand of human chromosome 7, in the region between base pairs 15047153-14824042, as denoted by the NCBI annotation. Peptides encoded by this sequence can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. Another embodiment of an NRP has been annotated, with a DNA sequence from the human genome located in the region 116668725-116667697 on the reverse strand of chromosome 6 (region according to NCBI human genome annotation project). The resulting peptide, NRP-6 (SEQ ID NO: 13), can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. Yet further embodiments of NRPs are found in rodents. A mouse NRP is encoded by a nucleic acid sequence located within the arachne contig_191157 of NCBI consisting of 339 nucleic acids using reading frame 1. Within an overlapping region, there is a second ORF of 198 nucleic acids starting at position 29 of an annotated NRP using frame 3. This ORF codes for a protein with high identity to a truncated human DNA repair protein. The resulting peptide, NRP-7 (SEQ ID NO: 17 can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. A still further embodiment is NRP-8 (SEQ ID NO:20), which is also a mouse peptide encoded by a nucleic acid sequence located within the genomic clone bM344E9 of the mouse Sanger database on the reverse strand. The protein coding sequence has been annotated and is located between base pairs 5609-4052. NRP-8 can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. A still further embodiment is NRP-9 (SEQ ID NO:28) is a rat orthologue NRP of the mouse NRP-7 (SEQ ID NO: 17) and is encoded by a nucleic acid sequence located on the reverse strand of rat chromosome 6 in the following exons: exon 1 located in position 7022614 - 7022326 and exon 2 located in position 7018685 - 7018651 (NCBI database). NRP-9 can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth. In another aspect, the invention includes embodiments for in vitro bioassays for evaluating proliferative and migration-inducing activity. Until recently, there were few in vitro neuronal migration assays available that could detect migrating untagged neurons over a prolonged time-period. One of these bioassays monitors olfactory peripheric placode cells organized as organotypic tissue cultures ("OTCs") during a 5-day time course (Fueshko and Wray, 1994). In certain embodiments of this invention, by using in vitro bioassay using adult thalamocortical OTCs," NRPs can be evaluated for their ability to induce neuronal migration, neuronal proliferation, neuronal differentiation, neuronal survival and/or neurite outgrowth. These embodiments can be particularly useful because 1) under control conditions, formation of a cell-bridge between both cultivated organs (e.g., thalamus and cortex) can be avoided by physically separating the two organs sufficiently far from each other (about 3 to about 5mm) on a tissue culture substrate and 2) because after birth, intrathalamic neuronal migration has been substantially completed due to the time course of thalamic ontogenesis. These bioassays can therefore be well suited for broad screening and identification of neuronal migration- inducing factors. In certain embodiments of an in vitro thalamocortical OTC assay includes the advantages of revealing both radial migration within the cortex and induced tangential migration within the thalamus. Under in vitro control conditions, only intrinsic cortical radial migration can be observed because of the normal time course of ontogenetic development of the neocortex. In other embodiments, in vitro bioassays are provided that involve cerebellar microexplants adhered to substrates. These embodiments can be used to provide data regarding patterns of neuronal migration, including quantifying the numbers of migrating neurons and the distance of migration in respect of the microexplant. A developing migration-chain consisting of small neurons (such as inhibitory granule cells) as well as an overall enhancement of cell migration can be observed after as little as 2-3 days of cultivation. This assay result resembles the cell chain induction within thalamocortical OTCs. Embodiments of another aspect of the invention include the use of NRPs to treat or prevent neurodegenerative diseases and brain injuries. In particular, NRPs are particularly suitable for use in brain regions lacking quiescent neuronal stem cells near the area of injury or disease. Use of NRPs as preventative agents can find use in elective surgeries, such as coronary artery bypass graft (CABG) procedures or other procedures involving a compromise of oxygen delivery to the brain. Moreover, NRPs can be useful in treating acute brain injuries caused by, for example, stroke, trauma or other injury that compromises oxygenation of the brain or spinal cord. Additionally, prophylactic treatment can be carried out before radiotherapy or chemotherapy. NRP compounds are capable of initiating neuronal proliferation, neuronal migration, neuronal survival and/or neurite outgrowth within postnatally differentiated neural tissue. These properties can be exploited in treatment strategies aimed at improving or repairing neuronal circuits within impaired areas of patients with mild to severe traumatic brain injury ("TBI"), including diffuse axonal injury, hypoxic-ischemic encephalopathy and other forms of craniocerebral trauma. NRP compounds can be used to treat infections of the nervous system, such as common bacterial meningitis, and to treat strokes including those caused by ischemic infarction, embolism and haemorrhage such as hypotensive haemorrhage or other causes. Moreover, NRP compounds can be useful for the treatment of neurodegenerative diseases including Alzheimer's disease, Lewy Body dementia, Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis, motor neuron disease, muscular dystrophy, peripheral neuropathies, metabolic disorders of the nervous system including glycogen storage diseases, and other conditions where neurons are damaged or destroyed. In certain embodiments of this invention, we found that members of the NRP gene family are expressed in mammalian stem cells, both in immortalized stem cells and in primary cultures of stem cells. In other embodiments, we found that NRPs can promote differentiation of stem cells into neural progenitor cells (neuroblasts). In still other embodiments, NRPs can stimulate migration of stem cells in response to chemoattractants, can promote differentiation of neuroblasts into cells having morphology of mature neurons (e.g., axons), and can promote the growth of neurites (e.g., axons and dendrites) from differentiated neuroblasts. Embodiments of other aspects of the invention include use of NRPs to increase proliferation of olfactory cells. Thus, NPRs can be important therapeutic tools to repair injured nerve cells, to cause repopulation of neural tissue, to aid in differentiation of neurons or to aid in processes necessary to promote synaptogenesis (e.g., neurite outgrowth and/or neural differentiation). In other embodiments, surgical implantation of stem cells in combination with an NRP can be used to repopulate neural tissues. The combination of stem cells or alternatively, neuroblast cells, along with an NRP can promote the regrowth of neural tissue. Such procedures can lead to reformation of mature neural tissues, and therefore can be used to treat neurodegenerative conditions. Such conditions including hypoxia/ischemia, stroke, cardiac graft bypass surgery, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and other disorders involving death or degeneration of neural cells. BRIEF DESCRIPTION OF THE FIGURES This invention will be described by way of description of particular embodiments thereof. Other objects, features and advantages of embodiments of this invention will become apparent from the specification and the figures, in which: Figure IA depicts the structure of the gene encoding NRP-7 with an intron (SEQ ID NO:35), highlighting the promoter region containing two CpG islands (one predicted for

NRP, the downstream one predicted for DNA-repair protein), as well as the existence of the

NRP gene as a 2bp frameshift of a known gene encoding a DNA repair protein. The primer positions for obtaining the NRP-7 (SEQ ID NO: 35) gene product are indicated. Figure IB shows 72.2% homology between the mouse (NRP-7 long) and the rat NRP (NRP-9) orthologues, the red number indicates the homology compared within the biological active NRP domains while the blue lines depict putative N-glycosylation sites. Figure 1C shows the alignment of human cachexia-related protein with mouse NRP

(NRP-7 long). Note the conservation of leucine, glycine and proline amino acid residues throughout both sequences. The overall homology is 34.4%. While the biological active domain of the cachexia-related protein is located directly after the signal peptide at position 20, the active domain of mouse NRP (NRP-7 long) is starting at amino acid position 83. Figure ID shows 52.5% overall homology between mouse NRP fragment and the human trefoil protein Ps2. Nine of fifteen amino acid residues that define the trefoil factor family (TFF) consensus sequence are present in NRPs, thereby confirming the occurrence of a trefoil factor domain in NRP. Figure IE shows the alignment between mouse SDF-1 a and mouse NRP (NRP-7 long) protein sequences. There is a moderate homology of 32.6% amongst both neuronal chemoattractive molecules. Figure 2 depicts survival induction by NRP-4 segment GQ (SEQ ID NO: 26) after excitotoxic/oxidative injury using 3-NP/glutamate. Figure 3 depicts proliferation induction with NRP-7 segment SW (SEQ ID NO: 24) in neurons injured using 3-NP/glutamate. Figure 4A depicts a haptotactic migration assay with mouse MEB-5 cells using lOng/ml of NRP-4 GG (SEQ ID NO:29) peptide for coating, with 200,000 MEB-5 cells. Figure 4B depicts results of a migration assay with primary mouse stem cells (E14) using lOng/ml of NRP-4 GG (SEQ ID NO:29) peptide for coating, with 200,000 cells seeded. Figure 4C depicts results of a migration assay with mouse MEB-5 cells using 1 ng/ml of NRP-2 KS (SEQ ID NO:23) peptide coating, with 200,000 cells seeded. Figure 4D depicts a migration assay with primary mouse stem cells using lOng/ml of NRP-7 SW (SEQ ID NO:24) coating, with 400,000 cells seeded. Figure 4E depicts migration assay with wild-type PC-12 cells using 50ng/ml of NRP-

4 GG (SEQ ID NO:29) coating, 200,000 cells per well. Figure 4F depicts a migration assay with wild type PC-12 cells and lng/ml NRP-7 SW (SEQ ID NO:24) coating followed by matrigel/PDL coating, with 100,000 cells seeded. In each case, the NRP promoted neuronal migration. Figures 5A and 5B depict graphs showing the medium length of the five longest axons per culture well of neural stem cells differentiated for 7 days in the presence of the NRPs. Figure 5A depicts effects of NRP-7 SW (SEQ ID NO:24) and IGF-1. Figure 5B depicts effects of NRP-2KS (SEQ ID NO:23), IGF-1 or differentiation medium alone. Significant differences from control medium were observed as determined by two-tailed Students t-test. ** pO.Ol; *** pθ.001. Figures 5C and 5D depict representative examples of longest axonal outgrowth in differentiation medium alone. Figure 5D depicts the longest axonal outgrowth in the presence of 10 nM NRP-7 SW (SEQ ID NO:24), whereas Figure 33D depicts the control condition. Figures 5E and 5F depict graphs showing the effects of NRP-7 SW (SEQ ID NO:24) and NRP-2KS (SEQ ID NO:23) on the increase in the of NSC cells that had undergone neuronal differentiation. Figure 5G indicates that nanomolar concentrations of NRP-2KS (SEQ ID NO:23) promoted the production of neuronal progenitors at the expense of multipotent stem cells from mouse forebrain neural stem cells. Figure 5H shows NRP-9 segment SD (SEQ ID NO:34) administration to undifferentiated NSCs before the onset of the differentiation experimental scheme. The upregulation of BrdU-positive cells under NRP treatment occurs while there is no obvious change in the overall cell number. Figures 6A-6K depicts gene expression of NRP within NSCs and in embryonic mouse tissues. Figures 6A-6C depict photographs of gels showing expression levels of mouse NRP (upper band, 412 bp) were semi-quantitatively compared to β-actin expression (lower band, 260 bp) in multiplex PCR. Figure 6A shows that mouse NRP expression was detected in embryonic brain tissue from El 5 mice in the cortex (Ctx), striatum (Str) and the olfactory bulb (OB). Albeit, the level was much lower compared to acutely isolated and immortalized mouse neural stem cells. Figure 6B shows differentiation of neural stem cells towards astrocytes with CNTF, markedly increases NRP mRNA expression, compared to undifferentiated stem cells, or neuronal differentiation with BDNF, Figure 6C depicts a gel showing analysis of a variety of El 9 embryonic mouse tissues shows that, except for the lung, expression of mouse NRP is lower, or absent in non nervous system tissues, compared with embryonic cortex, astrocytic differentiated NSC and astrocyte cultures from the perinatal forebrain. Figure 6D depicts a Northern blot hybridization with a 88 bp probe, non-overlapping with the DNA repair protein sequence, detecting two alternative mRNAs approximately sized 0.8 and 1.2 kb in RNA from perinatal astrocyte cultures. Figure 6E depicts in situ hybridization with the mouse NRP antisense probe labeling NSC. Figure 6F depicts lack of specific signal in the sense control. Figure 6G depicts NRP mRNA expression in NSC. Figure 6H depicts nestin localization to the same sites as NRP mRNA expression shown in Figure 6G. Figure 61 depicts in situ hybridization of NRP mRNA in coronal El 5 mouse forebrain slices, the ventricular zone, the cortical anlage, especially in the subplate and marginal zones and less intense in the cortical plate. Figure 6J depicts nestin positive cells spanning the length from the subplate to the marginal zone. Figure 6K depicts co-expression of mouse NRP message at the same sites as nestin expression in Figure 6J. Figure 6L depicts expression of the NRP-2 (SEQ ID NO:4) gene product in NT-2 cells in the absence of injury and 6 hrs post-injury caused by the mitochondrial toxin 3- nitroproprionic acid. Gene expression was substantially decreased when the cells were treated with 3-NP for more than 1 hr. Figures 7A and 7B depicts neuronal survival and proliferation induction by NRP-7 Segment SW. Figure 7A shows the effects of NRP-7SW (SEQ ID NO: 24) on neuroprotective activity over a broad dosage range of from 0.1 pM to 100 nM. Figure 7B shows that SDF-1 had only a limited neuroprotective effect compared to NRP. Figures 8 A and 8B depict results of studies of neuronal migration induction by NRPs. Figure 8A depicts a graph showing that rat and mouse synthetic peptide-derived NRPs (NRP-9SD and NRP-7SW) exhibited chemoattractive properties in attracting neuronal stem cells ("NSCs") as shown with a haptotactic migration assay. The efficacy of NRP-7SW was similar to that of SDF-1, but the NRPs displayed higher potency. Although the magnitudes of effects of SDF-1 and NRP-7SW appear similar, the amount of SDF-1 (100 nM) was higher than that of NRP-7SW (0.2 nM). Thus, NRP-7SW is about 500 times more potent than SDF-1. Figure 8B depicts a microphotograph of brain OTCs showing travelling medial ganglionic eminence-derived neural precursor cells migrating towards the cortical anlage. Figure 8C depicts a graph of quantification of the OTC assay demonstrating an maximal effect of NRP-7SW at a concentration of InM, whereas SDF-1 was less potent. Student's t-test was used for statistical analysis (* p<0.05, * * pO.Ol, % * % pO.OOl - N=6). Figures 9A - 9D depict results of expression and functional studies of full-length mouse recombinant NRP-7 long (SEQ ID NO:35). Figure 9A shows a Northern blot demonstrating that under the control of a cytomegalovirus ("CMV") promoter, mouse NRP-7 long (SEQ ID NO:35) gene 0.8kb signal was highly overexpressed in HEK cells, as detected by the NRP gene-specific 88bp-cRNA probe. Figure 9B is a Western blot showing that NRP-7 long (SEQ ID NO: 36) is expressed by HEK-cells and migrates at a molecular weight of 20kDa. Figure 9C depicts a graph showing that recombinant Myc-NRP-7 long (SEQ ID

NO:35)-HEK cells provide 51% neuroprotection to oxidative/excitotoxic-injured cerebellar microexplants. Figure 9D depicts a graph showing that Myc-NRP-7 long (SEQ ID NO:36)-HEK cells possess chemoattractive activity for attracting NSCs in a haptotactic migration assay when seeded on the bottom of the dish. More than twice the number of cells was attracted from the insert to the bottom of the culture dish compared to Myc-HEK control experiments. Student's t-test was applied for statistical analysis ( pO.OOl; N=4 for microexplants and * pO.OOl; N=6 for haptotactic migration assay). Figures 10A-10C depict results of studies using antibodies against CSCR4 and NRPs on P4-cerebellar explants. Figure 10A depicts a graph showing NRP-9 SD (SEQ ID NO:34) inhibition of neurite outgrowth. Figure 10B depicts NRP-2KS (SEQ ID NO:23) inhibition of neurite outgrowth. Chemoattractive effects of lOng/ml NRP coated on the culture dish were completely blocked by pre-incubating the neuronal stem cell line MEB-5 for 1.5hrs with a neutralizing antibody for CXCR4. Figure 10C shows that antibodies against CXCR4 reduce the chemoattractive effects of NRP-9SD compared to cells treated with NRP-9 SD (SEQ ID NO:34) alone. Figures 11A-11C depict results of studies using MAPK inhibitor on effects of NRPs, suggesting a possible signalling cascade involved in NRP neuroprotective and migration- inducing activity. Figure 11A depicts a graph showing that the MAPK (MEK)-inhibitor PD98509 completely blocked neuroprotective activity of rat NRP-9 SD (SEQ ID NO: 34) over a range of different NRP concentrations. Figure 11B depicts a graph showing that like PD98509, the PIK-3 inhibitor wortmannin inhibited the neuroprotective activity of NRP-9 SD (SEQ ID NO: 34). Figure 11C depicts a graph showing that the NRP-9 SD (4nM) induced increase in migrating NSC numbers was inhibited by 15 μM PD98509, whereas the basal migration level in BSA coated wells was not significantly altered by the MEK-inhibitor (% p<0.05, * * p<0.01, * * * pO.OOl - N=8). Figure 12A depicts results of studies in which NRP-4 segment PQ (SEQ ID NO:43) provided substantial neuroprotection in all analysed brain regions five days after insult when administered ICV 2hrs after hypoxia. Figure 12B depicts results of studies in which NRP-5 segment RG (SEQ ID NO: 30) administered ICV 2hrs after hypoxia provided substantial neuroprotection in all analysed brain regions five days after the hypoxic insult. Figure 13 depicts results of studies in which NRP-7 segment SW (SEQ ID NO:24) enhance proliferation of primary human adult olfactory ensheating glia. Figures 14A-14D depict results of studies of the neuroprotective activity of 4 NRP-5 RG analogues in reference to the original NRP-5 RG sequence. Figure 15 depicts a graph of proliferation-inducing effects of NRP-5 RG in embryonic cerebellar cells. Figure 16 depicts a graph of results of a haptotactic migration assay using mouse NSCs and NRP-5 RG as chemoattractant. Figure 17 depicts a graph of results of neuroprotective effects NRP-4 PQ after 48hrs of oxidative stress (0. ImM hydrogen peroxide). Figure 18 depicts a graph of neuroprotective effects of phosphorylated NRP-7 SW (NRP-7 ^PSW). Figure 19 depicts a graph of studies showing effects of NRP 9 SD on BrDU stained nuclei. Figures 20A and 20B depict micrographs of gene expression of NRP-7 (SEQ ID NO: 35) within the neuroepithelial stem cell - radial glia -astrocytic lineage. Figure 20A depicts expression of the mouse frameshift NRP transcript in the cerebral cortex of El 7 mouse brain, detected with the 88mer specific probe by in situ hybridization on cryosections. Figure 20B depicts a photomicrograph of the same section with an anti- vimentin antibody, demonstrating a high degree of co-localization of the mouse frameshift message with the vimentin intermediate filament protein. DETAILED DESCRIPTION

Definitions The term "homolog" includes one or more genes whose gene sequences are significantly related because of an evolutionary relationship, either between species (ortholog) or within a species (paralog). Homolog also includes genes related by descent from a common ancestral DNA sequence. Homolog also includes a relationship between genes separated by a speciation event, or to a relationship between genes by the event of genetic duplication (see paralog). As used herein, the term "homolog" also includes gene products related to each other by way of an evolutionary relationship. NRPs having conserved amino acid sequence domains are examples of homologs. The term "paralog" includes one of a set of homologous genes that have diverged from each other as a consequence of genetic duplication. For example, the mouse alpha globin and beta globin genes are paralogs. As used herein, the term "paralog" also includes gene products related to each other by way of an evolutionary relationship. Human NRPs having conserved amino acid sequence domains are examples of paralogs. The teπn "ortholog" includes one of a set of homologous genes that have diverged from each other as a consequence of speciation. For example, the alpha globin genes of mouse and chick are orthologs. As used herein, the term "ortholog" also includes gene products related to each other by way of an evolutionary relationship. Human and mouse

NRPs having conserved amino acid sequence domains are examples of homologs. The term "paralog peptide" includes a peptide encoded by a paralog nucleotide sequence. The term "peptide" and "protein" include polymers made of amino acids. The term "prodrug" includes molecules, including pro-peptides which, following enzymatic, metabolic or other processing, result in an active NRP, an active NRP analog or a NRP paralog. The tenn "NRP compound" includes NRPs, NRP homologs, NRP paralogs, NRP orthologs, NRP analogs, andprodrugs of NRP. The term "NRP" includes peptides having functions including one or more of neural migration, neuroblast migration, neural proliferation, neuronal differentiation, neuronal survival and neurite outgrowth, regardless of evolutionary relationship. Amino acids are represented by the standard symbols where alanine is represented by

"A" or "Ala", arginine by "R" or "Arg", asparagine by "N" or "Asn", aspartic acid by "D" or "Asp", cysteine by "C" or "Cys", glutamic acid by "E" or "Glu", glutamine by "Q" or "Gin", glycine by "G" or "Gly", histidine by "H" or "His", isoleucine by "I" or "He", leucine by "L" or "Leu", lysine by "K" or "Lys", methionine by "M" or "Met", phenylalanine by "F" or "Phe", proline by "P" or "Pro", serine by "S" or "Ser", threonine by "T" or Thr", tryptophan by "W" or "Trp", tyrosine by "Y" or "Tyr", and valine by "V" or "Val". Carboxy terminally amidated peptides are indicated by -NH₂. Nucleic acids comprise nucleotides including adenine, which is represented by "a"; thymine, which is represented by "t"; cytosine, which is represented by "c" and guanine, which is represented by "g." A nucleotide, which can be either guanine or adenine, is represented by "r", a nucleotide that can be either thymine or cytosine is represented by "y" and a nucleotide, which can be guanine, adenine, cytosine, or thymine is represented by "n". Polynucleotides may be DNA or RNA, and may be either single stranded or double stranded. Where the polynucleotide is a RNA polynucleotide, uracil "u" may be substituted for thymine. "Disease" includes any unhealthy condition of CNS or peripheral nervous system of an animal, including particularly Parkinson's disease, Lewy Body, Huntington's disease,

Alzheimer's disease, multiple sclerosis, motor neuron disease, muscular dystrophy, peripheral neuropathies, metabolic disorders of the nervous system including glycogen storage diseases. "Injury" includes any acute damage of an animal, including particularly stroke, traumatic brain injury, hypoxia, ischemia, perinatal asphyxia associated with fetal distress such as following abruption, cord occlusion or associated with intrauterine growth retardation, perinatal asphyxia associated with failure of adequate resuscitation or respiration, severe CNS insults associated with near miss drowning, near miss cot death, carbon monoxide inhalation, ammonia or other gaseous intoxication, cardiac arrest, coma, meningitis, hypoglycaemia and status epilepticus, episodes of cerebral asphyxia associated with coronary bypass surgery, hypotensive episodes and hypertensive crises, cerebral trauma and spinal cord injury.

Description of Specific Embodiments Embodiments of this invention include compositions and methods for the treatment of brain damage, encompassing neural regeneration peptides (NRPs). NRPs can induce neuronal migration, neurite outgrowth, neural differentiation, neural survival and/or neural proliferation. NRPs may be NRP analogs, paralogs, orthologs and/or NRP prodrugs, and peptides encoded by human, mouse or other species' genes. Some of the NRPs described herein are based on predicted protein sequence based upon the previously sequenced oligonucleotides corresponding to the genes noted herein. Other peptides are synthetic, and at least some are presented as a C-terminal amidated form. However, it can be appreciated that non-amidated forms of the proteins and peptides are to be included within the scope of this invention. The nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of rat NRP-l are:

9 18 27 36

5' tat gat cca gag gcc gcc tct gcc cca gga teg ggg Tyr Asp Pro Glu Ala Ala Ser Ala Pro Gly Ser Gly 45 aac cct tgc cat 3 ' SEQ ID NO: 1 Asn Pro Cys His-NH₂ SEQ ID NO: 2 Due to the degeneracy of the genetic code, however, multiple codons may encode the same amino acid. Thus, various nucleic acid sequences may encode for the same amino acid sequence. Each of these variations can be translated into SEQ ID NO: 2, and thus, all of these variations are included within the scope of this invention. For example, multiple nucleic acid sequences, including the nucleic acid sequence listed in SEQ ID NO: 1, encode for the rat NRP-l amino acid sequence. The invention further comprises variants of the nucleotide sequence of SEQ ID NO: 1, including variants which preserve the amino acid sequence encoded by the nucleic acid sequences, as well as nucleic acid sequences which encode for rat NRP-l analogs and NRP-l orthologs and/or paralogs. By way of example only, variants of SEQ ID NO: 1 according to the genetic code for DNA are listed below, with each codon separated by a space from neighbouring codons, and where a nucleic acid following a "/" is a variant for the nucleic acid preceding the "/":

5' tat/c gat/c cca/t/c/g gag/a gcc/g/a/t gcc/g/a t tct/a/c/g gcc/g/at cca/t/c/g gga/t/c/g tcg/a/t/c ggg/a t/c aac/t cct/a c/g tgc/t cat/c 3' The above sequence, including the indicated variants, may be written using the letters r, y and n as defined above to provide the following sequence:

5 ' tay gay ccn gar gen gen ten gen ccn ggn ten ggn aay ccn tgy cay 3 ' SEQ ID NO: 3 It will be understood that other nucleotide sequences encoding other NRPs can vary according to the redundancy of the genetic code. Moreover, RNA as well as DNA may encode the peptides of the invention, and that where a nucleic acid is a RNA nucleic acid, uracil may be substituted for thymine. A human gene was annotated using the human cachexia cDNA (US Patent No: 5,834,192) as a template. A survival-promoting peptide has more than 96% identity to a survival-promoting peptide (Cunningham et al., 1998) and rat NRP-l has 100% identity to the cachexia protein and is the only NRP-l homologue with known respective cDNA. Human cachexia protein is localised on chromosome 12 within the region of base pairs 621841- 625428 and consists of 5 exons. We have compared the cachexia mRNA splice sites with the identified NRP human paralog on chromosome 13 (genomic clone from the Sanger Sequencing Centre - bA87Gl: base pairs 77232-76768) and have annotated the coding region of a NRP-l human ortholog (this ortholog is herein termed NRP-2). The nucleotide and amino acid sequences relating to NRP-2 are:

SEQ ED NOs: 4 and 5 9 18 27 36

5' atg aga gtc aga gta caa etc aag tct aat gtc caa gtt gga Met Arg Val Arg Val Gin Leu Lys Ser Asn Val Gin Val Gly 45 54 63 72 81 gca gga cac tea gca aag gat cca gag gca agg aga gca cct Ala Gly His Ser Ala Lys Asp Pro Glu Ala Arg Arg Ala Pro 90 99 108 117 126 gga age eta cat ccc tgt eta gca gca tea tgc tea get get Gly Ser Leu His Pro Cys Leu Ala Ala Ser Cys Ser Ala Ala 135 144 153 162 ggc ctg cac aca age teg tgg aag aac ctg ttt ttg ata gaa Gly Leu His Thr Ser Ser Trp Lys Asn Leu Phe Trp lie Glu 171 180 189 198 207 gga eta gta agt att tgc eta ggg cac ata gtt gta caa gag Gly Leu Val Ser He Cys Leu Gly His He Val Val Gin Glu 216 225 234 243 252 acg gac gtt ttt agg tec ttg egg ttt ctt gca ttt cca gaa Thr Asp Val Phe Arg Ser Leu Arg Phe Leu Ala Phe Pro Glu 261 270 279 288 aac ttg ctt caa ata ttt ttc cag atg caa aat tec ttg gat Asn Leu Leu Gin He Phe Phe Gin Met Gin Asn Ser Leu Asp 297 306 315 324 330 cct tgt ttt aga atg aat eta tta aaa act tea cat taa 3 ' SEQ ID NO:4 Pro Cys Phe Arg Met Asn Leu Leu Lys Thr Ser His *stop SEQ ID NO:5 The underlined nucleotide sequence denotes the signal peptide. The protein-encoding DNA sequence consists of 4 exons as predicted by splice site analysis taking the sequence of the paralog form of the human cachexia gene (cDNA from US patent 5,834,192) on chromosome 12 as a template. The chromosome map of the genomic clone bA87Gl is considered as the basis for the exact exon localisation. Exon 1 is located between bp 77232-77170. Exon 2 is located between bp 77088-77046. Exon 3 is located between bp 77036-76824. Exon 4 is located between base pairs 76778-76768 followed by the translation stop codon TAA. The translated protein consists of 110 amino acids, is identical in length to the human cachexia protein, and has 24.5% overall identity to human cachexia protein. Sequence comparison of the signal peptides for extracellular localisation (amino acids 1-19) of both proteins reveals 31.6% identity. Significantly, comparison of the first 30 amino acids of the mature (cleaved) peptide reveals 46.7% amino acid identity. Furthermore this peptide has similar neuronal migration, proliferation, survival and neurite outgrowth activities as NRP-l (see Figures 16, 17 and 18). A second ortholog of NRP-l has been annotated, and is encoded by a DNA sequence from the human genome located between the base pairs 34764-33003 on the reverse complement strand of chromosome 3 (region according the Double Twist human genome annotation project). The protein coding sequence consists of 5 exons with the following locations: exon 1: 34764-34743; exon 2: 34729-34700; exon 3: 33745-33596; exon 4: 33498- 33459; exon 5: 33043-33003. The nucleotide sequence (SEQ ID NO: 6) has 333 nucleotides and the amino acid sequence (SEQ ID NO: 7; herein termed NRP-3) has 111 amino acids, as denoted below.

SEQ ID NOs: 6 and 7 9 18 27 36

5' atg aaa ata aat gta tta att aaa tta atg ace aag tea gat Met Lys He Asn Val Leu He Lys Leu Met Thr Lys Ser Asp 45 54 63 72 81 tct ttt aaa age caa gcc agg ggc caa gtt ccc cca ttt eta Ser Phe Lys Ser Gin Ala Arg Gly Gin Val Pro Pro Phe Leu 90 99 108 117 126 999999 gtg 999 tg ^ccc t99 ttt ttt caa aca agg ttt tgg Gly Gly Val Gly Cys Pro Trp Phe Phe Gin Thr Arg Phe Trp 135 144 153 162 ggc cat agt ttt gca gtt aaa ctg gcc tec aac ctt tec cag Gly His Ser Phe Ala Val Lys Leu Ala Ser Asn Leu Ser Gin

171 180 189 198 207 gca gag aaa ttg gtc ctt cag caa ace ctt tec caa aaa ggc Ala Glu Lys Leu Val Leu Gin Gin Thr Leu Ser Gin Lys Gly 216 225 234 243 252 eta gac gga gca aaa aaa get gtg ggg gga etc gga aaa eta Leu Asp Gly Ala Lys Lys Ala Val Gly Gly Leu Gly Lys Leu 261 270 279 288 gga aaa gat gca gtc gaa gat eta gaa age gtg ggt aaa gga Gly Lys Asp Ala Val Glu Asp Leu Glu Ser Val Gly Lys Gly 297 306 315 324 333 gcc gtc cat gac gtt aaa gac gtc ctt gac tea gta eta tag 3' SEQIDNO:6 Ala Val His Asp Val Lys Asp Val Leu Asp Ser Val Leu *stop SEQIDNO:7 These sequences belong to the human gene family of NRPs, and it is herein termed

NRP-3. The sequence has 50% identity and 62.7% similarity to the human cachexia- associated protein. Furthermore, the peptide encoded by this nucleotide sequence has similar properties to NRP-l. A third NRP-l ortholog has been annotated is contained in the DNA sequence from the human genome located between the region 21970003-21972239 on the forward strand of human chromosome 15 (region according NCBI human genome annotation project). The protein coding sequence consists of 6 exons with the following locations: exon 1: 21970003- 21970031; exon 2: 21970515-21970545; exon 3: 21970571-21970644; exon 4: 21970818- 21970861; exon 5: 21971526-21971731; exon 6: 21972189-21972239. This gene has been re-sequenced and now is believed to be reflected in SEQ ID NO:48 and SEQ ID NO:49 below. However, the resequencing of the gene has not altered the NRP encoded thereby. The sequence consists of 435 nucleic acids that encode 145 amino acids. The nucleotide sequence

(SEQ ID NO: 8) and predicted amino acid sequence (SEQ ID NO: 9; herein termed NRP-4) are:

SEQ ID NO: 8 and 9 9 18 27 36

5' atg get gtt gtg tta ctt gca cca ttt ggg gac ate age cag Met Ala Val Val Leu Leu Ala Pro Phe Gly Asp He Ser Gin 45 54 63 72 81 gaa ate aca aag gtt ggg aca ggg act cca ggg agg get gag Glu He Thr Lys Val Gly Thr Gly Thr Pro Gly Arg Ala Glu 90 99 108 117 126 gcc ggg ggc cag gtg tct cca tgc ctg gcg gcg tec tgc agt Ala Gly Gly Gin Val Ser Pro Cys Leu Ala Ala Ser Cys Ser 135 144 153 162 cag gcc tat ggc gcc ate ttg get cac tgc aac etc tgc etc Gin Ala Tyr Gly Ala He Leu Ala His Cys Asn Leu Cys Leu 171 180 189 198 207 cca ggt tea atg att aaa aaa aag aag aaa ttt ata gtt gaa , Pro Gly Ser Met He Lys Lys Lys Lys Lys Phe He Val Glu 216 225 234 243 252 ata gaa agt caa cct tta aag tct tac agg gaa aat tct ace He Glu Ser Gin Pro Leu Lys Ser Tyr Arg Glu Asn Ser Thr 261 270 279 288 cat ttt ccc aga cca gtc eta aat ctt atg cga aaa cac tgt His Phe Pro Arg Gly Val Leu Asn Leu Met Arg Lys His Cys 297 306 315 324 333 ggg gaa aag ggg gaa gaa ggg cct tgt ttc tct ccc aag caa Gly Glu Lys Gly Glu Glu Gly Pro Cys Phe Ser Pro Lys Gin 342 351 360 369 378 atg ggg gag agg cga gnn tgt ggc gga ggg eta ggg ttg get Met Gly Glu Arg Arg XXX Cys Gly Gly Gly Leu Gly Leu Ala 387 396 405 414 cgc gag ate act aat tta aca tec get cat ctg ttg gtc ttg Arg Glu He Thr Asn Leu Thr Ser Ala His Leu Leu Val Leu 423 432 435 aat ate age aac cag tga 3 ' SEQ fD NO:8 Asn He Ser Asn Gin *stop SEQ ID NO:9 This sequence belongs to the human gene family NRPs. This sequence has 45% amino acid similarity to the NRP encoded by a nucleic acid sequence located on human chromosome 13. Triplet 244-246 (amino acid position 82); triplet 391-393 (amino acid position 131) and triplet 421-423 (amino acid position 141) encode potential N-glycosylation sites. Amino acid position 118 has an x because of uncertainty within the nucleic acid sequence. The peptide, NRP-4, exhibits neural proliferation promoting activity, neurite outgrowth and neuronal survival promoting activities. Note that in oligonucleotide position 353-354, the nucleotide had not been determined and the corresponding amino acid is not known. However, subsequent to the publication of the above sequence, a correct sequence has been provided and is described herein as Example 26, SEQ ID NO: 48 and SEQ ED NO:49. We note that the change in sequence information does not appear to affect the sites relevant to neuroprotective effects of peptides derived from SEQ ID NO:8 or SEQ ID NO:9. I Another human ortholog ("NRP-5") of rat NRP-l is encoded by the DNA sequence located within the Homo sapiens chromosome 7 working draft (NCBI: ref/NT _007933.9/Hs7_8090) of the NCBI database on the reverse strand. The protein coding sequence has been annotated and consists of 3 exons with 798 nucleic acids in total length coding for 266 amino acids. The exact locations for the protein coding exons are the following: exon 1: 15047153-15046815; exon 2: 14897885-14897772; exon 3: 14824386- 14824042. There exists evidence from a human EST (GenBank AW138864) that the mRNA is expressed. The nucleotide sequence (SEQ ID NO: 10) and the amino acid sequence (SEQ ID NO: 11 ; NRP-5) are as follows:

SEQ ED NOs: 10 and 11 9 18 27 36

5' atg ctg gac ccg tct tec age gaa gag gag teg gac gag ggg Met Leu Asp Pro Ser Ser Ser Glu Glu Glu Ser Asp Glu Gly 45 54 63 72 81 ctg gaa gag gaa age cgc gat gtg ctg gtg gca gcc ggc age Leu Glu Glu Glu Ser Arg Asp Val Leu Val Ala Ala Gly Ser 90 99 108 117 126 teg cag cga get cct cca gcc ccg act egg gaa ggg egg egg Ser Gin Arg Ala Pro Pro Ala Pro Thr Arg Glu Gly Arg Arg 135 144 153 162 gac gcg ccg ggg cgc gcg ggc ggc ggc ggc gcg gcc aga tct Asp Ala Pro Gly Arg Ala Gly Gly Gly Gly Ala Ala Arg Ser 171 180 189 198 207 gtg age ccg age ccc tct gtg etc age gag ggg cga gac gag Val Ser Pro Ser Pro Ser Val Leu Ser Glu Gly Arg Asp Glu 216 225 234 243 252 ccc cag egg cag ctg gac cat gag cag gag egg agg ate cgc Pro Gin Arg Gin Leu Asp Asp Glu Gin Glu Arg Arg He Arg 261 270 279 288 ctg cag etc tac gtc ttc gtc gtg agg tgc ate gcg tac ccc Leu Gin Leu Tyr Val Phe Val Val Arg Cys He Ala Tyr Pro 297 306 315 324 333 ttc aac gcc aag cag ccc ace gac atg gcc egg agg cag cag Phe Asn Ala Lys Gin Pro Thr Asp Met Ala Arg Arg Gin Gin 342 351 360 369 378 aag ctt aac aaa caa cag ttg cag tta ctg aaa gaa egg ttc Lys Leu Asn Lys Gin Gin Leu Gin Leu Leu Lys Glu Arg Phe 387 396 405 414 cag gcc ttc etc aat ggg gaa ace caa att gta get gac gaa Gin Ala Phe Leu Asn Gly Glu Thr Gin He Val Ala Asp Glu 423 432 441 450 459 gca ttt tgc aac gca gtt egg agt tat tat gag gtt ttt eta Ala Phe Cys Asn Ala Val Arg Ser Tyr Tyr Glu Val Phe Leu 468 477 486 495 504 aag agt gac cga gtg gcc aga atg gta cag agt gga ggg tgt Lys Ser Asp Arg Val Ala Arg Met Val Gin Ser Gly Gly Cys 513 522 531 540 tct get aag gac ttc aga gaa gta ttt aag aaa aac ata gaa Ser Ala Asn Asp Phe Arg Glu Val Phe Lys Lys Asn He Glu 549 558 567 576 585 aaa cgt gtg egg agt ttg cca gaa gtg gat ggc ttg age aaa Lys Arg Val Arg Ser Leu Pro Glu He Asp Gly Leu Ser Lys 594 603 612 621 gag aca gtg ttg age tea tgg ata gcc aaa tat gat gcc att Glu Thr Val Leu Ser Ser Trp He Ala Lys Tyr Asp Ala He 630 639 648 657 666 tac aga ggt gaa gag gac ttg tgc aaa cag cca aat aga atg Tyr Arg Gly Glu Glu Asp Leu Cys Lys Gin Pro Asn Arg Met

675 684 693 702 711 gcc eta agt gca gtg tct gaa ctt att ctg age aag gaa caa Ala Leu Ser Ala Val Ser Glu Leu He Leu Ser Lys Glu Gin 720 729 738 747 etc tat gaa atg ttt cag cag att ctg ggt att aaa aaa ctg Leu Tyr Glu Met Phe Gin Gin He Leu Gly He Lys Lys Leu 756 765 774 783 792 gaa cac cag etc ctt tat aat gca tgt cag| gta agt ggt etc Glu His Gin Leu Leu Tyr Asn Ala Cys Gin Val Ser Gly Leu 798 tga 3' SEQ IDNO:10 *stop SEQIDNO:ll The entire protein NRP-5 consists of 266 amino acids. The annotated translated NRP amino acid sequence NRP-5 has 76% similarity to a human calcium dependent activator protein of secretion (GenBankXP_036915) located on chromosome 3. Furthermore, exon 1 (339 nucleic acids) of the translated human chromosome 7 NRP-5 has 95.5% homology to a translated mouse 5' EST (PJKENBB632392). This protein shares domains present in NRP-l and other NRPs that exhibit biological properties of neurite outgrowth, neuronal survival, neuronal proliferation and neuronal migration. We have annotated a DNA sequence from the human genome located between the regions 116668725-116667697 on the reverse complement strand of chromosome 6 (region according NCBI human genome annotation project). The protein coding sequence consists of 3 exons with the following locations: exon 1: 116668725-116668697, exon 2: 116668333- 116668305, and exon 3: 116667872-116667697. The sequence, herein termed NRP-6 consists of 234 nucleic acids that encode 78 amino acids. This sequence belongs to the human gene family of NRPs. The highest homology found to human ESTs presents identity from nucleic acids 59-234 compared to the human cDNA clone GenBankCS0DK001YI19 isolated from human placental tissue. This clone was sequenced from the 3 '-prime end and consists of 924 nucleic acids. Because our homologue form ends with the stop codon TGA after 234 nucleic acids we are not dealing with the same gene product. The nucleotide sequence (SEQ ID NO: 12) encoding for an NRP, and the amino acid sequence (SEQ ID NO: 13; NRP-6) for the peptide is:

SEQ ID NOs: 12 and 13 9 18 27 36 5 ^l atg aga gac aaa caa cat eta aat gca aga cat aaa aag gaa Met Arg Asp Lys Gin His Leu Asn Ala Arg His Lys Lys Glu 45 54 63 72 81 agg aag gag aga tea tat agt aca aca eta caa ggt gtt etc Arg Lys Glu Arg Ser Tyr Ser Thr Thr Leu Gin Gly Val Leu 90 99 108 117 126 aac aaa aag tct ttg tta gac ttc aat aat act att tgg tac Asn Lys Lys Ser Leu Leu Asp Phe Asn Asn Thr He Trp Tyr 135 144 153 162 ttc tat cag caa ata gga age att cca ata ctt att aga tec Phe Tyr Gin Gin He Gly Ser He Pro He Leu He Arg Ser 171 180 189 198 207 tct ace ate aga cac aga aat tac eta gaa aac aga aat gta Ser Thr He Arg His Arg Asn Tyr Leu Glu Asn Arg Asn Val 216 225 234 ttg cca aat etc aaa caa gag ggc tga 3' SEQ IDNO:12 Leu Pro Asn Leu Lys Gin Glu Gly *stop SEQIDNO:13 The amino acid sequence of NRP-6 has 14.1% identity and 44.9% similarity to the annotated NRP paralog on human chromosome 13, NRP-2. This protein shares domains present in NRP-l and other NRPs (e.g., NRPs 2-5) that have biological properties of neurite outgrowth, neuronal survival, neuronal proliferation and neuronal migration. Furthermore, another NRP-l ortholog has been identified, a mouse NRP family member. The mouse NRP family member (here indicated as protein 2, SEQ ID NO: 17; herein termed NRP-7) is located within the arachne contig_191157 of NCBI consisting of 339 nucleic acids using reading frame 1. Within an overlapping region there is a second ORF of

198 nucleic acids starting at position 29 of the annotated NRP paralog using frame 3. This

ORF codes for a protein (here indicated as protein 1) with high identity to a truncated human

DNA repair protein. By using the search paradigm tBLASTN using the biological active NRP peptide sequence: KDPEARRAPGSLHPCLAASCSAAG-NH₂ (SEQ ID NO: 18) we got a blast hit in the mouse EST RIKEN database. This 5 '-generated mouse EST has the accession number GenBankAK012518 and the following sequence (SEQ ID NO: 14):

5'- GGCAGCCTCGAGATGGGGAAGATGGCGGCTGCTGTGGCTTCATTAGCCACGCTG GCTGCAGAGCCCAGAGAGGATGCTTTCCGGAAGCTTTTCCGCTTCTACCGGCAGA GCCGGCCGGGGACAGCGGACCTGGGAGCCGTCATCGACTTCTCAGAGGCGCACT TGGCTCGGAGCCCGAAGCCCGGCGTGCCCCAGGTAGGAAAGGAGGAGTAGTGTG TGCCAGCCTAGCGGCCGACTGGGCCACCCGAGACTGGGCCGCCTCCGGGCCGGC TTTGGAGGGAAGCCCCTGCTGGGCCTGTCCAGTGAGCTGTAATGTCGAGCGATGA GCGACCAGCTGCCTCGCTGTCCCAACGCTCTGGCCACGGCTTGTGCCTTGCCGCC ATTTCCCCCAACCCACGCGGGCCACGGCTTGTGCCCTGCCGCCATTTCCCCCAAC CCACGCGACCTTGCTC - 3 ' SEQ ID NO: 14

Protein 1 Reading Frame 3 Translation of open reading frame 3 (ORF of 198 nucleic acids starting at position 13 of the EST) reveals the following protein sequence (SEQ ID NO: 15): MGKMAAAVASLATLAAEPREDAFRKLFRFYRQSRPGTADLGAVIDFSEAHLARSPK PGVPQVGKEE SEQ ID NO: 15 This sequence has 82% homology (identity and chemical similarity) of amino acid sequence to the human alkylated DNA repair protein with the GenBank accession number Q13686. The mouse form is C-terminal truncated and has only 66 of the 389 amino acids of the human DNA repair protein.

Protein 2 Reading Frame 1 An even longer ORF of 323 nucleic acids can be found within frame 1 of the EST sequence. We then annotated the 5' end of the 323 nucleic acid ORF in the mouse genome and found a new gene located in the mouse arachne contig_191157 sequence of the NCBI database between 23970 and 24374. The protein coding sequence consists of two exons with an overall length of 339 nucleic acids coding for 113 amino acids. The location of exon 1 is: 23970-23990, and for exon 2 it is: 24057-24374. The nucleotide sequence (SEQ ID NO: 16) and the amino acid sequence (SEQ ID NO:17; NRP-7) of this mouse NRP ortholog of rat NRP-l are: SEQ H) NOs: 16 and 17 9 18 27 36 5' atg aat cga aac cct gga gtc cct cga gat ggg gaa gat ggc Met Asn Arg Asn Pro Gly Val Pro Arg Asp Gly Glu Asp Gly 45 54 63 72 81 ggc tgc tgt ggc ttc att age cac get ggc tgc aga gcc cag Gly Cys Cys Gly Phe He Ser His Ala Gly Cys Arg Ala Gin

90 99 108 117 126 aga gga tgc ttt ccg gaa get ttt ccg ctt eta ccg gca gag Arg Gly Cys Phe Pro Glu Ala Phe Pro Leu Leu Pro Ala Glu 135 144 153 162 ccg gcc ggg gac age gga cct ggg age cgt cat cga ctt etc Pro Ala Gly Asp Ser Gly Pro Gly Ser Arg His Arg Leu Leu 171 180 189 198 207 aga ggc gca ctt ggc teg gag ccc gaa gcc egg cgt gcc cca Arg Gly Ala Leu Gly Ser Glu Pro Glu Ala Arg Arg Ala Pro 216 225 234 243 252 ggt agg aaa gga gga gta gtg tgt gcc age eta gcg gcc gac Gly Arg Lys Gly Gly Val Val Cys Ala Ser Leu Ala Ala Asp 261 270 279 288 tgg gcc ace cga gac tgg gcc gcc tec ggg ccg get ttg gag Trp Ala Thr Arg Asp Trp Ala Ala Ser Gly Pro Ala Leu Glu 297 306 315 324 333 gga age ccc tgc tgg gcc tgt cca gtg age tgt aat gtc gag Gly Ser Pro Cys Trp Ala Cys Pro Val Ser Cys Asn Val Glu 339 cga tga 3 ' SEQ ID NO: 16 Arg *stop SEQ ID NO: 17 The entire expressed amino acid sequence of NRP-7 contains 113 amino acids (SEQ ID NO: 17). An alternative version of NRP-7 is an alternatively spliced form containing an additional 66 nucleotides after position 21 of SEQ ID NO: 16 (SEQ ID NO:35), which produces a long form of NRP-7 ("NRP-7 long") having 135 amino acids (SEQ ID NO:36). 9 18 27 36 5' ATG AAT CGA AAC CCT GGA GTC GTG ACC CCG GAA GAA CCT GCC Met Asn Arg Asn Pro Gly Val Val Thr Pro Glu Glu Pro Ala 45 54 63 72 81 AGA GCC GGA ATT TCG AGT TCT GCT TCC GGG CCA AAC TGT TGG Arg Ala Gly He Ser Ser Ser Ala Ser Gly Pro Asn Cys Trp

90 99 108 117 126 CAG CCT CGA GAT GGG GAA GAT GGC GGC TGC TGT GGC TTC ATT Gin Pro Arg Asp Gly Glu Asp Gly Gly Cys Cys Gly Phe He 135 144 153 162 AGC CAC GCT GGC TGC AGA GCC CAG AGA GGA TGC TTT CCG GAA Ser His Ala Gly Cys Arg Ala Gin Arg Gly Cys Phe Pro Glu 171 180 189 198 207 GCT TTT CCG CTT CTA CCG GCA GAG CCG GCC GGG GAC AGC GGA Ala Phe Pro Leu Leu Pro Ala Glu Pro Ala Gly Asp Ser Gly 216 225 234 243 252 CCT GGG AGC CGT CAT CGA CTT CTC AGA GGC GCA CTT GGC TCG Pro Gly Ser Arg His Arg Leu Leu Arg Gly Ala Leu Gly Ser 261 270 279 288 GAG CCC GAA GCC CGG CGT GCC CCA GGT AGG AAA GGA GGA GTA Glu Pro Glu Ala Arg Arg Ala Pro Gly Arg Lys Gly Gly Val

297 306 315 324 333 GTG TGT GCC AGC CTA GCG GCC GAC TGG GCC ACC CGA GAC TGG Val Cys Ala Ser Leu Ala Ala Asp Trp Ala Thr Arg Asp Trp 342 351 360 369 378 GCC GCC TCC GGG CCG GCT TTG GAG GGA AGC CCC TGC TGG GCC Ala Ala Ser Gly Pro Ala Leu Glu Gly Ser Pro Cys Trp Ala

387 396 405 408 3' TGT CCA GTG AGC TGT AAT GTC GAG CGA TGA (*stop) SEQ ID NO:35 Cys Pro Val Ser Cys Asn Val Glu Arg SEQ ID NO:36 The protein function program tool SMART predicts a signal peptide sequence consisting of 28 amino acids. The protein has 13.6% identity and 23.6% similarity towards the NRP ortholog on human chromosome 13, and has neuronal survival, migration, proliferation and outgrowth activity similar to NRP-l . A second mouse NRP family member is located within the genomic clone bM344E9 of the mouse Sanger database on the reverse complement strand. By using the search program tBLASTN using the biologically active NRP peptide sequence: KDPEARRAPGSLHPCLAASCSAAG-NH₂ (SEQ ID NO: 18) we obtained an area of similarity in the genomic mouse Sanger database within the genomic clone bM344E9. The protein coding sequence has been annotated and consists of 5 exons and is 423 nucleic acids in total length coding for 141 amino acids. The locations for the coding exons are the following: exon 1: 5609-5596, exon 2: 5502-5489, exon 3: 5398-5283, exon 4: 5243-5229, and exon 5: 5215-4952. The coding nucleotide sequence (SEQ ID NO: 19) and the amino acid sequence (SEQ ID NO:20) of the mouse ortholog of rat NRP-l (herein termed NRP-8) is:

SEQ ID NOs: 19 and 20 9 18 27 36 5 ' atg tgc act ctg cag gta tgg tct tec tec etc cct tec etc Met Cys Thr Leu Gin Val Trp Ser Ser Ser Leu Pro Ser Leu 45 54 63 72 81 ccc cac etc tct gag ggg tea ggg gtc age att tgg atg ctg Pro His Leu Ser Glu Gly Ser Gly Val Ser He Trp Met Leu 90 99 108 117 126 etc cca cca ggc cca get tta gaa atg aat tec tec ggc etc Leu Pro Pro Gly Pro Ala Leu Glu Met Asn Ser Ser Gly Leu 135 144 153 162 ctt tat act ctt gag ace tec tgg gga ace agg ace etc ttg Leu Tyr Thr Leu Glu Thr Ser Trp Gly Thr Arg Thr Leu Leu 171 180 189 198 207 get cct ctg gtg aca tac atg gga tct gat gca tct gag gtg Ala Pro Leu Val Thr Tyr Met Gly Ser Asp Ala Ser Glu Val 216 225 234 243 252 gat gca aga aga gca aaa aag agt etc cac tgc ate ctg tct Asp Ala Arg Arg Ala Lys Lys Ser Leu His Cys He Leu Ser 261 270 279 288 gac ace age cat ccc egg ggc cat gcc egg aat gag agg agg Asp Thr Ser His Pro Arg Gly His Ala Arg Asn Glu Arg Arg 297 306 315 324 333 ctt ggc ctt ggg gtt tgg aag ace gag ctt tgg gtc cag ace Leu Gly Leu Gly Val Trp Lys Thr Glu Leu Trp Val Gin Thr 342 351 360 369 378 ctg eta tea ctg atg gtg aca tec tgg gaa gtt tat gaa act Leu Leu Ser Leu Met Val Thr Ser Trp Glu Val Tyr Glu Thr 387 396 405 414 cgt teg tgc etc agt ttc ccc ate agg cct tta get cac tgg Arg Ser Cys Leu Ser Phe Pro He Arg Leu Leu Ala His Trp 423 gga taa 3 ' END SEQ ID NO:19 Gly *stop SEQ ID NO:20 The expressed amino acid sequence of NRP-8 contains 141 amino acid residues. The asparagine residue at position 112-114 is putatively N-glycosylated according to the occurrence of an N-glycosylation consensus sequence. The new mouse NRP-l ortholog NRP-l 0 has 35.5% homology to the human NRP ortholog located on chromosome 13 (NRP- 2) and 28.9% homology to the mouse NRP-l ortholog located on the arachne contig from NCBI. Furthermore this peptide comprises amino acid sequence domains similar to those present in NRP-l or other NRP peptides and this peptide has biological properties including promotion of neuronal migration, proliferation, survival and/or neurite outgrowth. The nucleotide sequence (SEQ ID NO:27) and the amino acid sequence (SEQ ID

NO:28) of NRP-9, the rat ortholog of mouse NRP-7 is: SEQ ID NOs: 27 and 28 1 9 18 27 36 45 ATG TTA AAA CTG AAT GAA CCA AAG CCT GGG GTC GTG ACC TCG GAA Met Leu Lys Leu Asn Glu Pro Lys Pro Gly Val Val Thr Ser Glu 54 63 72 81 90 GAA CTT ACA GGA TCC GGA ATT TGG AGT TCT GCT TCC GGG CCA AAC Glu Leu Thr Gly Ser Gly He Trp Ser Ser Ala Ser Gly Pro Asn 99 108 117 126 135 TGT TCG CAA CAT CGA GAT GGG GAA GAT GGC GGC TGC GGT CGT TTC Cys Ser Gin His Arg Asp Gly Glu Asp Gly Gly Cys Gly Arg Phe 144 153 162 171 180 ATT AAC CTC GCT GGC AAC AGA ACC CAA AGA GGA TGC TTT CCG GAA He Asn Leu Ala Gly Asn Arg Thr Gin Arg Gly Cys Phe Pro Glu 189 198 207 216 225 GCT TTT CCG CTT CTA CAG GCA GAG CCG GCG GAG TAC GGC GGA CCT Ala Phe Pro Leu Leu Gin Ala Glu Pro Ala Glu Tyr Gly Gly Pro 234 243 252 261 270 AGG AGC GGT CAT CGA CTT CTC AGA GGC TCA CGT GGC TCA GAG CCC Arg Ser Gly His Arg Leu Leu Arg Gly Ser Arg Gly Ser Glu Pro 279 288 297 306 315 GAA GCC CGG CGT GCC CAA GGT GGT CAG ATT CCC TCT GAA CGT GTC Glu Ala Arg Arg Ala Gin Gly Gly Gin He Pro Ser Glu Arg Val 324 CTC AGT GAC TGA SEQ ID NO:27 Leu Ser Asp stop SEQ ID NO:28

In addition to the NRP compounds described above, we have identified other genes having NRP-like peptide domains that also can be useful for expressing NRPs. These include genes from mycobacteria and rumor cells. A recently published paper has disclosed a PE multigene family of Mycobacterium tuberculosis containing a consensus sequence (PE_PGRS) that is similar to our proposed sequence (PGR/S). They also mention that these proteins are released in the host, by the bacterium, to promote bacterial survival. Here are the examples they provided in the paper, where the PE_PGRS consensus sequence was found. The amino acid sequence of the Rvl818c gene product of M. tuberculosis (SEQ ID NO:21) is shown below: MSFWTIPEA LAAVATDLAG IGSTIGTANA AAAVPTTTVL AAAADEVSAA MAALFSGHAQ AYQALSAQAA LFHEQFVRAL TAGAGSYAAA EAASAAPLEG VLDVINAPAL ALLGRPLIGN GANGAPGTGA NGGDGGILIG NGGAGGSGAA GMPGGNGGAA GLFGNGGAGG AGGNVASGTA GFGGAGGAGG LLYGAGGAGG AGGRAGGGVG GIGGAGGAGG NGGLLFGAGG AGGVGGLAAD AGDGGAGGDG GLFFGVGGAG GAGGTGTNVT GGAGGAGGNG GLLFGAGGVG GVGGDGVAFL GTAPGGPGGA GGAGGLFGVG GAGGAGGIGL VGNGGAGGSG GSALLWGDGG AGGAGGVGST TGGAGGAGGN AGLLVGAGGA GGAGALGGGA TGVGGAGGNG GTAGLLFGAG GAGGFGFGGA GGAGGLGGKA GLIGDGGDGG AGGNGTGAKG GDGGAGGGAI LVGNGGNGGN AGSGTPNGSA GTGGAGGLLG KNGMNGLP SEQ ID NO:21

The amino acid sequence of Epstein-Barr Virus Nuclear Antigen 1 (SEQ ID NO:22) is shown below:

MSDEGPGTGP GNGLGEKGDT SGPEGSGGSG PQRRGGDNHG RGRGRGRGRG GGRPGAPGGS GSGPRHRDGV RRPQKRPSCI GCKGTHGGTG AGAGAGGAGA GGAGAGGGAG AGGGAGGAGG AGGAGAGGGA GAGGGAGGAG GAGAGGGAGA GGGAGGAGAG GGAGGAGGAG AGGGAGAGGG AGGAGAGGGA GGAGGAGAGG GAGAGGAGGA GGAGAGGAGA GGGAGGAGGA GAGGAGAGGA GAGGAGAGGA GGAGAGGAGG AGAGGAGGAG AGGGAGGAGA GGGAGGAGAG GAGGAGAGGA GGAGAGGAGG AGAGGGAGAG GAGAGGGGRG RGGSGGRGRG GSGGRGRGGS GGRRGRGRER ARGGSRERAR GRGRGRGEKR PRSPSSQSSS SGSPPRRPPP GRRPFFHPVG EADYFEYHQE GGPDGEPDVP PGAIEQGPAD DPGEGPSTGP RGQGDGGRRK KGG FGKHRG QGGSNPKFEN IAEGLRALLA RSHVERTTDE GT VAGVFVY GGSKTSLYNL RRGTALAIPQ CRLTPLSRLP FGMAPGPGPQ PGPLRESIVC YFMVFLQTHI FAEVLKDAIK DLVMTKPAPT CNIRVTVCSF DDGVDLPPWF PPMVEGAAAE GDDGDDGDEG GDGDEGEEGQ E SEQ ID NO:22

From Brennan, MJ. and Delogu, G.,(2002). The PE multigene family: a 'molecular mantra' for mycobacteria. Trends in Microbiology 5: 246-249. It can be appreciated that the entire sequence of NRP-l - NRP 9 need not be used.

Rather, peptide fragments of about 8 amino acids can be used according to embodiments of this invention. Given the consensus sequence domains herein identified, one can fashion synthetic peptides or can truncate naturally occurring NRPs to obtain portions of peptides that are biologically active. Methods of truncation (e.g., using synthetic DNA) or enzymatic modification of expressed peptides are known in the art. One embodiment of the invention is a 24-mer fragment of NRP-2 (SEQ ID NO: 5) comprising the sequence KDPEARRAPGSLHPCLAASCSAAG-NH₂ (NRP-2 segment KG;

SEQ ID NO: 18). Another embodiment of the invention is a 19-mer fragment of NRP-2 (SEQ ID NO:

5) comprising the sequence KDPEARRAPGSLHPCLAAS-NH₂ (NRP-2 segment KS; SEQ

ID NO: 23). Yet another embodiment of the invention is a 24-mer form of NRP-7 (SEQ ID NO:

17 or SEQ ID NO: 36) comprising the sequence SEPEARRAPGRKGGWCASLAADW-

NH₂ (NRP-7 segment SW; SEQ ID NO: 24). Further embodiment of the invention is an 11-mer peptide comprising the sequence SDSFKSQARGQ-NH₂ (NRP-3 segment SQ; SEQ ID NO:25), located between amino acids 13-23 of NRP-3 (SEQ ID NO:7). Another embodiment of the invention is an 11-mer peptide comprising sequence GTPGRAEAGGQ-NH₂ (NRP-4 segment GQ; SEQ ID NO: 26), located between amino acids 22-32 of the annotated NRP-4 (SEQ ID NO:9). Another embodiment of the invention is a 25-mer fragment of NRP-4 (SEQ ID NO: 9) comprising sequence GTPGRAEAGGQVSPCLAASCSQAYG-NH₂ (NRP-4 segment GQ; SEQ ID NO: 29) Yet another embodiment of the invention is a 13-mer fragment of NRP-5 (SEQ ID

NO: 11) comprising sequence REGRRDAPGRAGG-NH₂ (NRP-5 segment RG; SEQ ID NO: 30). Still further embodiment of the invention is a 24-mer fragment of NRP-8 (SEQ ID NO 20) comprising sequence SEVDARRAKKSLHCILSDTSHPRG-NH₂ (NRP-8 segment SG; SEQ ID NO: 31) Yet another embodiment of the invention is a 24-mer fragment of NRP-3 (SEQ ID NO: 7) comprising sequence SDSFKSQARGQVPPFLGGVGCPWF-NH₂ (NRP-3 segment SF; SEQ ID NO: 32) Another embodiment of the invention is an 8-mer fragment of NRP-5 (SEQ ID NO: 11) comprising sequence REGRRDAP-NH₂ (NRP-5 RP; SEQ ID NO: 33). Additional embodiment of the invention is a 21-mer peptide comprising sequence SEPEARRAQGGQIPSERVLSD-NH₂ (NRP-9 segment SD; SEQ ID NO: 34), which is located between amino acid residues 88-108 of NRP-9 (SEQ ID NO: 28). Another embodiment of the invention is a 9-mer peptide comprising sequence PGRAEAGGQ-NH₂ (NRP-4 segment PQ; SEQ ID NO: 43), located between amino acids 24- 32 of the annotated NRP-4 (SEQ ID NO:9). Further embodiments are described elsewhere herein.

Uses of NRP Compounds Thus, the invention includes embodiments which relate to NRPs, peptides encoded by

NRPs, homologs, orthologs or paralogs of NRPs, analogs of NRPs, and prodrugs of NRPs, where a prodrag of an NRP is a molecule that may be enzymatically, metabolically or otherwise modified to become an NRP, a NRP homolog, NRP paralog, an NRP ortholog or an NRP analog. Such molecules are collectively termed as "NRP compounds" or "NRPs." NRP compounds may be encoded for by nucleotide sequences, which may be DNA or RNA and which may be single stranded or double stranded. It will be understood that the invention includes sequences complementary to the sequences described in this application as well as the sequences themselves. It is also to be understood that there may be alternatively spliced forms of NRPs, in which case, those alternatively spliced forms of NRP RNA, and the proteins and peptides they may encode are also considered to be part of this invention. As indicated above, embodiments of the present invention are based upon the inventors' surprising finding that NRPs can induce neurons and neuroblasts to proliferate, migrate, differentiate, produce neuritis and can protect neurons against damage caused by neural insults. Proliferation and migration of neural cells into areas of damage caused by acute brain injury or chronic neurodegenerative disease can result in improvement in neural functioning. Further, NRPs can promote neuronal survival, neuronal differentiation, and/or neurite outgrowth. Thus, NRP compounds may be used to treat a variety of disorders and conditions where brain tissue degenerates, is at risk of degeneration or death, or has died. Cells can also use NRP oligonucleotides to stimulate production of NRPs after transfection. In some cases, transfection can be in a replicable vehicle, and in others, the NRP oligonucleotide can be introduced as naked DNA.

Disorders and Conditions Treatable with NRPs Disorders and conditions in which NRP compounds can be of benefit include: Infections of the central nervous system including bacterial, fungal, spirochetal, parasitic and sarcoid including pyrogenic infections, acute bacterial meningitis, leptomeningitis; Cerebrovascular diseases including stroke, ischemic stroke, atherosclerotic thrombosis, lacunes, embolism, hypertensive haemorrhage, ruptured aneurysms, vascular malformations, transient ischemic attacks, intracranial haemorrhage, spontaneous subarachnoid haemorrhage, hypertensive encephalopathy, inflammatory diseases of the brain arteries, decreased perfusion caused by, for example, cardiac insufficiency (possibly resulting from coronary bypass surgery) and other forms of cerebrovascular disease; Craniocerebral trauma including basal skull fractures and cranial nerve injuries, carotid-cavernous fistula, pneumocephalus, aerocele andrhinorrhea, cerebral contusion, traumatic intracerebral haemorrhage, acute brain swelling in children; Demyelinating diseases include neuromyelitis optica, acute disseminated encephalomyelitis, acute and subacute necrotizing haemorrhagic encephalitis, diffuse cerebral sclerosis of Schilder and multiple sclerosis in conjunction with peripheral neuropathy. Degenerative diseases of the nervous system including syndrome of one or more of progressive dementia, diffuse cerebral atrophy, diffuse cortical atrophy of the non-Alzheimer type, Lewy body dementia, Pick's disease, fronto-temporal dementia, thalamic degeneration, non-Huntingtonian types of Chorea and dementia, cortico-spinal degeneration (Jakob), the dementia-Parkinson-amyotrophic lateral sclerosis complex (Guamanina and others); Acquired metabolic disorders of the nervous system including metabolic diseases presenting as a syndrome comprising one or more of confusion, stupor or coma-ischemia- hypoxia, hypoglycaemia, hyperglycemia, hypercapnia, hepatic failure and Reye syndrome, metabolic diseases presenting as a progressive extrapyramidal syndrome, metabolic diseases presenting as cerebellar ataxia, hyperthermia, celiac-sprue disease, metabolic diseases causing psychosis or dementia including Cushing disease and steroid encephalopathy, thyroid psychosis and hypothyroidism, pancreatic encephalopathy; Diseases of the nervous system due to nutritional deficiency; Alcohol and alcoholism; Disorders of the nervous system due to drugs and other chemical agents including opiates and synthetic analgesics, sedative hypnotic drugs, stimulants, psychoactive drugs, bacterial toxins, plant poisons, venomous bites and stings, heavy metals, industrial toxins, anti-neoplastic and immunosuppressive agents, thalidomide, aminoglycoside antibiotics (ototoxicity) and penicillin derivatives (seizures), cardioprotective agents (beta-blockers, digitalis derivatives and amiodarone). As illustrated by the preceding list, compositions and methods of the invention can find use in the treatment of human neural injury and disease. Still more generally, the compositions and methods of the invention find use in the treatment of human patients suffering from neural damage as the result of acute brain injury, including but not limited to diffuse axonal injury, perinatal hypoxic-ischemic injury, traumatic brain injury, stroke, ischemic infarction, embolism, and hypertensive haemorrhage; exposure to CNS toxins, infections of the central nervous system, such as, bacterial meningitis; metabolic diseases such as those involving hypoxic-ischemic encephalopathy, peripheral neuropathy, and glycogen storage diseases; or from chronic neural injury or neurodegenerative disease, including but not limited to Multiple Sclerosis, Lewy Body Dementia, Alzheimer's disease, Parkinson's disease and Huntington's disease. Patient's suffering from such diseases or injuries may benefit greatly by a treatment protocol able to initiate neuronal proliferation and migration, as well as neurite outgrowth. Still more generally, the invention has application in the induction of neuronal and neuroblast migration into areas of damage following insult in the form of trauma, toxin exposure, asphyxia or hypoxia-ischemia. NRP compounds, including NRP-l, its orthologs, analogs, paralogs and prodrugs containing the identified NRP peptide domains, can be used to promote neuronal and neuroblast migration. Most conveniently, this can be affected through direct administration of NRP compounds to the patient. However, while NRPs can be advantageously used, there is no intention to exclude administration of other forms of NRP compounds. For example, human paralog forms or peptide fragments of NRP can be administered in place of NRP. By way of example, the effective amount of NRP in the CNS can be increased by administration of a pro-drug form of NRP that comprises NRP and a carrier, NRP and the carrier being joined by a linkage that is susceptible to cleavage or digestion within the patient. Any suitable linkage can be employed which will be cleaved or digested to release NRP following administration. Another suitable treatment method is for NRP levels to be increased through an implant that is or includes a cell line that is capable of expressing NRP or analogs, paralogs or pro-peptides of an NRP in an active form within the central nervous system of the patient. An NRP can be administered as part of a medicament or pharmaceutical preparation. This can involve combining NRP compounds with any phannaceutically appropriate carrier, adjuvant or excipient. Additionally an NRP compound can be used with other non-NRP neuroprotective, proliferative, or other agent. The selection of the carrier, adjuvant or excipient will of course usually be dependent upon the route of administration to be employed. The administration route can vary widely. An NRP may be administered in different ways: intraperitoneal, intravenous or intracerebroventricular. The peripheral application may be the way of choice because then there is no direct interference with the central nervous system. Any peripheral route of administration known in the art can be employed. These can include parenteral routes for example injection into the peripheral circulation, subcutaneous, intraorbital, ophthalmic, intraspinal, intracisternal, topical, infusion (using eg. slow release devices or minipumps such as osmotic pumps or skin patches), implant, aerosol, inhalation, scarification, intraperitoneal, intracapsular, intramuscular, intranasal, oral, buccal, pulmonary, rectal or vaginal. The compositions can be formulated for parenteral administration to humans or other mammals in therapeutically effective amounts (eg. amounts which eliminate or reduce the patient's pathological condition) to provide therapy for the neurological diseases described above. One route of administration includes subcutaneous injection (e.g., dissolved in 0.9% sodium chloride) and oral administration (e.g., in a capsule). It will also be appreciated that it may on occasion be desirable to directly administer

NRP compounds to the CNS of the patient. This can be achieved by any appropriate direct administration route. Examples include administration by lateral cerebroventricular injection or through a surgically inserted shunt into the lateral cerebroventricle of the brain of the patient. Determining Doses of NRP The determination of an effective amount of an NRP to be administered is within the skill of one of ordinary skill in the art, and will be routine to those persons skilled in the art. In certain embodiments, the amount of an NRP to be used can be estimated by in vitro studies using an assay system as described herein. The final amount of an NRP to be administered will be dependent upon the route of administration, upon the NRP used and the nature of the neurological disorder or condition that is to be treated. A suitable dose range may for example, be between about 0.01 mg to about 1 mg per 100 g of body weight, alternatively about 0.06 μg to about 0.6 mg of NRP-l per lOOg of body weight where the dose is administered centrally. For inclusion in a medicament, NRP can be directly synthesized by conventional methods such as the stepwise solid phase synthesis method of Merryfield et al, 1963 (J. Am. Chem. Soc. 15:2149-2154). Such methods of peptide synthesis are known in the art, and are described, for example, in Fields and Colowick, 1997, Solid Phase Peptide Synthesis (Methods in Enzymology, vol. 289), Academic Press, San Diego, CA. Alternatively synthesis can involve the use of commercially available peptide synthesizers such as the Applied Biosystems model 430 A. As a general proposition, the total pharmaceutically effective amount of NRP-l (SEQ ID NO: 2) administered parenterally per dose will be in a range that can be measured by a dose response curve. One range is between about 0.06 mg and about 0.6 mg per 100 g body weight. For example, NRP-l (SEQ ID NO:2) in the blood can be measured in body fluids of the mammal to be treated to determine dosing. Alternatively, one can administer increasing amounts of the NRP-l (SEQ ID NO:2) compound to the patient and check the serum levels of the patient for NRP-l (SEQ ID NO:2). The amount of NRP-l (SEQ ID NO:2) to be employed can be calculated on a molar basis based on these serum levels of NRP-l (SEQ ID NO:2). Specifically, one method for determining appropriate dosing of the compound entails measuring NRP levels in a biological fluid such as a body or blood fluid. Measuring such levels can be done by any means, including RIA and ELISA. After measuring NRP levels, the fluid is contacted with the compound using single or multiple doses. After this contacting step, the NRP levels are re-measured in the fluid. If the fluid NRP levels have fallen by an amount sufficient to produce the desired efficacy for which the molecule is to be administered, then the dose of the molecule can be adjusted to produce maximal efficacy. This method can be carried out in vitro or in vivo. This method can be carried out in vivo, for example, after the fluid is extracted from a mammal and the NRP levels measured, the compound herein is administered to the mammal using single or multiple doses (that is, the contacting step is achieved by administration to a mammal) and then the NRP levels are remeasured from fluid extracted from the mammal. NRP compounds are suitably administered by a sustained-release system. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, for example, films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), poly(2-hydroxyethyl methacrylate)

(Langer et al, 1981), ethylene vinyl acetate (Langer et al, supra), or poly-D-(-)-3- hydroxybutyric acid (EP 133,988). Sustained-release compositions also include a liposomally associated compound. Liposomes containing the compound are prepared by methods known to those of skill in the art, as exemplified by DE 3,218,121; Hwang et al, 1980; EP 52,322;

EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008, U.S. Pat.

Nos. 4,485,045 and 4,544,545 and EP 102,324. In some embodiments, liposomes are of the small (from or about 200 to 800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the most efficacious therapy. All U.S. parents refened to herein, both supra and infi-a, are hereby incorporated by reference in their entirety. PEGylated peptides having a longer life than non-PEGylated peptides can also be employed, based on, for example, the conjugate technology described in WO 95/32003 published November 30, 1995. For parenteral administration, doses may be between about 0.01 to about 1 mg per lOOg of body weight, alternatively about 0.06μg to 0.6 mg of NRP compound per lOOg body weight. In some embodiments, the compound can be formulated generally by mixing each at a desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically, or parenterally, acceptable carrier, i.e., one that is non- toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides. It can be appreciated that the above doses are not intended to be limiting. Other doses outside the above ranges can be determined by those with skill in the art. In some embodiments, formulations can be prepared by contacting a compound uniformly and intimately with liquid earners or finely divided solid carriers or both. Then, if desired, the product can be shaped into the desired formulation. In some embodiments, the canier is a parenteral carrier, alternatively, a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, a buffered solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein. The earner suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are desirably non-toxic to recipients at the dosages and concentrations employed, and include, by way of example only, buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpynolidone; glycine; amino acids such as glutamic acid, aspartic acid, histidine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, trehalose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counter-ions such as sodium; non-ionic surfactants such as polysorbates, poloxamers, or polyethylene glycol (PEG); and/or neutral salts, e.g., NaCl, KC1, MgCl₂, CaCl₂, etc. An NRP compound can be desirably formulated in such vehicles at a pH of from about 4.5 to about 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of salts of the compound. The final preparation may be a stable liquid or lyophilized solid. In other embodiments, adjuvants can be used. Typical adjuvants which may be incoφorated into tablets, capsules, and the like are a binder such as acacia, com starch, or gelatin; an excipient such as microcrystalline cellulose; a disintegrating agent like com starch or alginic acid; a lubricant such as magnesium stearate; a sweetening agent such as sucrose or lactose; a flavoring agent such as peppermint, wintergreen, or cherry. When the dosage form is a capsule, in addition to the above materials, it may also contain a liquid carrier such as a fatty oil. Other materials of various types may be used as coatings or as modifiers of the physical form of the dosage unit. A syrup or elixir may contain the active compound, a sweetener such as sucrose, preservatives like propyl paraben, a coloring agent, and a flavoring agent such as cherry. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice. For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occuning vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants, and the like can be incoφorated according to accepted pharmaceutical practice. Desirably, an NRP compound to be used for therapeutic administration may be sterile. Sterility can be readily accomplished by filtration through sterile filtration membranes (e.g., membranes having pore size of about 0.2 micron). Therapeutic compositions generally can be placed into a container having a sterile access port, for example an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. In other embodiments, an NRP compound can be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized fonnulation, 10-mL vials are filled with 5 ml of sterile-filtered 0.01% (w/v) aqueous solution of compound, and the resulting mixture is lyophilized. The infusion solution can be prepared by reconstituting lyophilized compounds using bacteriostatic water or other suitable solvent.

Gene Therapy In other embodiments of this invention, therapeutic methods include gene therapy for treating an organism, using a nucleic acid encoding an NRP compound. Generally, gene therapy can be used to increase (or overexpress) NRP levels in the organism. Examples of nucleotide sequences include SEQ ED NOs: 1, 3, 4, 6, 8, 10, 12, 16, 19, 27 or 35 or portions thereof that encode peptides having the consensus domains and biological properties of NRP. It can be appreciated that other sequences can be used to encode a pro-NRP, which, upon cleavage, can result in a biologically active NRP. Any suitable approach for transfecting an organism with a sequence encoding an NRP can be used. For example, in vivo and ex vivo methods can be used. For in vivo delivery, a nucleic acid, either alone or in conjunction with a vector, liposome, precipitate etc. can be injected directly into the organism, for example, a human patient, and in some embodiments, at the site where the expression of an NRP compound is desired. For ex vivo treatinent, an organism's cells are removed, the nucleic acid is introduced into these cells, and the modified cells are administered to the organism either directly or, for example, encapsulated within porous membranes which are implanted into the patient. See, e.g. U.S. Pat. Nos: 4,892,538 and 5,283,187. We have demonstrated herein that cultured cells can express NPRs, and that when those NRP-expressing cells are incubated with neurons susceptible to toxic damage, NPRs can be expressed, secreted into the medium and can protect the neurons from toxic damage. This suφrising finding supports a therapeutic approach to treating neural degeneration by gene transfer and cell transplantation. There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviras. In certain embodiments, in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, Heφes simplex I virus, or adeno-associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are N-[l-(2,3- dioleyloxy)propyl] -N,N,N-trimethylammonium (DOTMA), dioleoylphatidylethanolamine (DOPE) and 3-β[N-(N',N'-dimethylamionethane)carbomoyl]cholesterol (DC-Choi), for example. In some situations it may be desirable to provide the nucleic acid source with an agent that directs the nucleic acid-containing vector to target cells. Such "targeting" molecules include antibodies specific for a cell-surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins that bind to a cell-surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake. Examples of such proteins include capsid proteins and fragments thereof tropic for a particular cell type, antibodies for proteins, which undergo internalization in cycling, and proteins that target intracellular localization and enhance intracellular half-life. In other embodiments, receptor-mediated endocytosis can be used. Such methods are described, for example, in Wu et al, 1987 or Wagner et al, 1990. For review of the currently known gene marking and gene therapy protocols, see Anderson 1992. See also WO 93/25673 and the references cited therein. Kits are also contemplated within the scope of this invention. A typical kit can comprise a container, in some embodiments a vial, for the NRP formulation comprising one or more NRP compounds in a pharmaceutically acceptable buffer and instractions, such as a product insert or label, directing the user to utilize the pharmaceutical fonnulation.

EXAMPLES The following examples are provided to illustrate certain embodiments of this invention. It can be readily appreciated that other embodiments can be devised and still remain within the scope of this invention. All of these other embodiments are considered to be part of this invention.

Example 1: NRP Gene Identification To identify mammalian neuronal migration-inducing factors with efficacy on neuronal survival, proliferation and neuronal differentiation, we under took a screen of the rat and mouse genome using the human cachexia related protein cDNA and its encoded 16-mer cachexia fragment as a template to identify novel NRP homologues. Identification of the NRP genes involved obtaining total RNA from different cell sources (in vivo tissue, neural stem cell cultures). RNA was extracted using the Roche Total RNA Isolation Kit. Complementary DNA (cDNA) was synthesised using Superscript RT II, followed by multiplex PCR amplification of the mouse NRP gene fragment and beta-actin using the following primers: mouFS NRP Fwd primer: 5' AACGGAATGAATCGAAACCC 3' (SEQ ID NO:37); mouFS NRP Rev primer: 5' CGCTCGACATTACAGCTCA 3' (SEQ ED NO:38); mouse beta-actin Fwd: 5' GAAAGGGTGTAAAACGCAGC 3' (SEQ ID NO:39); Mouse B-actin Rev: 5' GGTACCACCATGTACCCAGG 3' (SEQ ID NO:40). The correct sized fragments were gel purified, cloned directly into pGEM vectors and transformed into competent strain of E. Coli (DH5α). The transformed cell colonies were screened for the presence of the NRP gene fragment, and the plasmids from positive colonies were sequenced The stracture of the gene encoding the NRP gene is depicted in Figure IA). Characterisation of NRP Protein Domains NRP is probably secreted over the non-classical pathway like FGF-1, FGF-2 because of highly significant scores when inteφreting the protein sequence using the SecretomeP server (Technical University of Denmark). The high number of positive amino acid residues and overall atom number within N-terminal NRP led to this prediction (Bendtsen et al., 2004). A single trefoil domain sequence motif can be predicted within the mouse NRP sequence and this domain has 52.5% homology (Figure ID) to human pS2 protein (Jakowlew et al., 1984). The putative NRP trefoil domain has 9 of 15 conserved amino acids within the consensus sequence of the trefoil domain (database SMART) as similar or identical. This 60% consensus value is also the threshold value for the acceptance of NRP as a trefoil factor family member. Trefoil domains have been implicated to participate in protein-protein interactions and acting through cyclooxygenase2 (COX-2) and thromboxane A2 receptor (TXA2-R) activation pathways (Rodrigues et al., 2003). The human Ps2 protein belongs to trefoil factor family 1 (TFF-1) and has been implicated in chemoattraction of breast cancer cells (Prest et al., 2002) by signalling over the ERK1/2 pathway (Graness et al., 2002). The neuronal survival, proliferation, migration and differentiation-promoting mouse

NRP (NRP-7 segment SW) domain is located C-terminal from the trefoil domain and is located at the C-terminus for rat NRP-9 (see aligned 21mer and 24mer sequences in Figure IB). Homology Between NRP, Cachexia-Related Protein and SDF-1 In spite of the existence of striking similar biological activities between NRPs and SDF-lαboth peptides reveal only a moderate similarity of 32.6% homology (Figure IE). It has been shown that the first 9 amino acids of mature SDF-1 display residual chemoattractive activity, approximately a factor 100 lower than SDF-lα (Loetscher et al., 1998). The N-terminal 11 amino acids of the mouse 24mer peptide (NRP-7 Segment SW;

SEQ ID NO:24) align with more than 50% homology to the start of mature SDF-1. The related protein domains indicate that the cachexia protein, NRPs and SDF-1 α share biological active domains that are important for neuronal survival, proliferation, migration and differentiation. For the cachexia-related protein and SDF-1 the biological active regions are situated N-terminal of the mature proteins while for the NRP this region is located C-terminal from the trefoil domain.

Example 2: Cerebellar Microexplants: Neuroprotection and Neuronal Proliferation Methods Laminated cerebellar cortex was extracted from P3/4, P7/8 rat pups and triturated through gauze having a 125 μm pore size to obtain uniformly sized microexplants. After centrifugation for 3 minutes at 61 g and the pellet was resuspended in StartV medium (Biochrom) and the suspension seeded on poly-D-lysine coated coverslips in 6-well plates and incubated for 3 hrs to allow adherence, before 1 ml StartV per well was added. As described previously, glutamate/3-NP and NRP were also added. The explants are cultivated at 34°C at 5% C0₂ and 100% humidity for 48-72 hrs. BrdU was administered at start of cultivation for proliferation rate measurements and cells are counted per microscopic field after 48-72 hrs.

Results (a) Neuroprotection Nanomolar concentrations of NRP-4 segment GQ (SEQ ID NO: 26), produced survival rates of 50% after severe injury (Figure 2). (b) Neuronal Proliferation NRP-7 segment SW (SEQ ID NO: 24) enhanced the proliferation rate in these cultures by more than 200% (Figure 3). Proliferative cells of cerebellar microexplants were not susceptible towards excitotoxic and oxidative stress compared to effects of vehicle alone.

Example 3: Haptotactic Migration Assays NRPs were tested for migration-inducing/chemoattractive activity on mouse neural stem cells, EGF-dependent immortalized mouse neural stem cell line MEB5 and wild-type PC-12 cells in a haptotactic migration assay as described below.

Methods Initial NRP Coating: Control wells of Transwell plates (Corning) with 12μm pore size were coated in 1.5ml of the BSA/PBS vehicle. Remaining plates were coated using various concentrations of NRPs ranging between l-100ng/ml (prepared in PBS containing lOug/ml BSA). The plates were then incubated at 37°C for lhr to coat. Wells were then rinsed 2x with 1ml sterile PBS.

Extracellular Matrix Coating: Laminin (10/xg/ml) for MEB-5 cells, PDL (50μg/ml) + Matrigel for mouse primary stem cells and fibronectin (25ug/ml) + matrigel for PC-12 cells were used as extracellular matrix (ECM) coating for the cells. All ECM compounds were diluted in PBS. 1.5ml of the ECM per well was incubated for 2hrs at room temperature. The wells were then rinsed once with 1ml serum-free media (e.g. NB/B27) followed by 1ml PBS wash. Coating of Inserts: A 5ug/mL PDL/PLL mixture (in PBS) was used to coat inserts. Subsequently the inserts were rinsed with MilliQ water. Transferring to Media and Cell Seeding: Appropriate medium (MEB-5 cells: DMEM-high glucose + N2 (growth medium supplement from Invitrogen) + lOng/ biotin +

2mM L-glutamine, primary stem cells: NSA-medium from Euroclone and for PC-12 cells:

NB/B27 medium) was transfened into the 12-well plates. The plates were then incubated at

37°C; 5% C0₂ and seeded with 1 - 2 x IO⁵ cells. Plates were fixed at 1-2 days in vitro (DP7). Fixation: Inserts were discarded and wells fixed in successive dilutions of PFA (0.4, 1.2, 3 and 4%) for 3-5min in each dilution. The wells were rinsed and stored in successive dilutions of PFA (0.4, 1.2, 3 and 4%) 3-5 min in each dilution. The wells were rinsed and stored in PBS until counting. All cells that displayed neurite outgrowth and traveled to the bottom chamber were counted as migrating cells. Results lOng ml NRP-4 GG (SEQ ID NO:29) caused 195% more MEB-5 cells to migrate to the bottom of the culture dish in comparison with the BSA-vehicle alone (Figure 4A). In the presence of NRP-4 GG (SEQ ID NO:29) peptide 93.7% more E14 cells migrated compared with the BSA-vehicle alone (Figure 4B). 109% more MEB-5 cells migrated to the bottom in the presence of NRP-2 KS (SEQ ID NO:23) compared with the BSA-vehicle alone (Figure 4C). 35% more E-14 cell density occuned in the presence of NRP-7 SW (SEQ ID NO:24) - treated wells compared with the BSA-vehicle (Figure 4D). 80.8% more PC-12 cells migrated to the bottom in the presence of NRP-4 GG (SEQ ID NO:29) peptide compared with the BSA vehicle alone (Figure 4E). NRP-7 SW (SEQ ED NO:24) caused 333% more PC-12 cells to migrate in compared with the BSA vehicle alone (Figure 4F). Example 4: Neural Stem Cell Culture and Differentiation Assay for Axonal Outgrowth NSA stem cell culture medium was purchased from Euroclone, Italy. Neurobasal medium, DMEMF12 medium, N2 and B27 supplement were all from Life Technology. Anti- βlϋ-tubulin antibody was purchased from Sigma. The Cy3 -conjugated goat-anti mouse antibody was purchased from Amersham and Syto21 from Molecular Probes. NRP-2KS (SEQ ID NO:23) and NRP-7 SW (SEQ ID NO:24) were used. Human recombinant Erythropoietin (EPO) was purchased from R&D Systems. Neural Stem Cell Culture Neural stem cells were derived from El 5 C3H mice forebrain and cultured as neurospheres in the presence of 20ng/ml EGF and lOng/ml bFGF as described in Gritti et al. (2001). Briefly, timed pregnant mice were sacrificed and the embryos removed. Under sterile conditions the brains were removed and the forebrains dissected. The tissue was dissociated by trituration and pelleted by 75g centrifugation for 10 minutes and the cells seeded in non- coated tissue culture flasks in NSA medium with growth factors. For passaging neurospheres were triturated and seeded as single cells. To determine whether NRPs could induce a shift of cells within neurospheres from symmetric (two stem cells, both stem cells) to asymmetric division of neural stem cells (1 stem cell, 1 neuronal progenitor or neuroblast cell), as has been described for erythropoietin (EPO) by Shingo et al. (2001), neurospheres were dissociated by trituration and seeded for expansion at a density of 200,000 cells/well in 6 well plates in NSA medium with EGF only, EGF plus varying concentrations of an NRP, or EGF plus 10 U/ml EPO. One half of the medium with fresh NRP compound was exchanged every other day. After 7 days the cells from the neurospheres were plated. In the presence of EGF, cells retain symmetric cell division. With the removal of EGF, cells begin to differentiate (asymmetric cell division). Subsequently, the cells were subjected to a differentiation assay as described below, with the exception that bFGF was not added to the plating medium for the initial 24 h and differentiation for 7 days was allowed in control medium without NRPs or other compounds. In cells that had never been exposed to an NRP, the numbers of neuroblast cells were lower than in cells that had been exposed to an NRP or EPO. Therefore, NRPs can increase the differentiation of undifferentiated stem cells into neuroblast cells.

Differentiation Assay Upon reaching a sufficient size, neurospheres were dissociated by trituration and plated at a density of 200,000 cells per well on laminin-coated 13mm diameter coverslips that had been placed in Nunc 24 well plates. The plating medium was a 1:1 mixture of DMEM/F12 supplemented with N2 and Neurobasal supplemented with B27 and 2mM glutamine. To enhance survival after plating the medium contained 2ng/ml bFGF. After 24h the medium was replaced with a neuronal differentiation-promoting medium (1:3 mixture of DMEM/F12 supplemented with N2 and Neurobasal supplemented with B27 and 2 mM glutamine) and except for the controls the test compound or IGF-1 was added simultaneously. The medium with fresh compound was exchanged every other day. Seven days after plating the differentiating cells were incubated for 20 minutes in differentiation medium containing lOOng/ml Syto21 to label the nuclei of viable cells. Subsequently, they were fixed with 4% paraformaldehyde and immunostained with a mouse anti-BIII tubulin antibody and a goat anti-mouse Cy3 -coupled secondary antibody.

Quantification of Neuronal Differentiation and Axonal Outgrowth After staining the coverslips were removed from the tissue culture plate and mounted on coverslips, using Immunofluore fluorescent mounting medium. To analyse the percentage of neurons of total cells, with a Zeiss axiophot microscope, equipped with Axoivision software, at 20x magnification images of two random fields per well were taken in the red (tubulin stained neurons) and green (Syto21 stained nuclei) fluorescent channel. Neurons and nuclei per field were counted and the neuronal percentage of total cell number detennined. Results of these studies are shown in Figures 5A-5H.

Results Over a wide concentration range NRP-7 segment SW increased the percentage of neuronal progeny from NSC, plated on laminin in differentiation medium when normalising the neuronal cell number to the total viable cell number within the differentiation assay. At a concentration of 10 pM a maximum of 2-fold increase in beta-III-tubulin-positive neurons was observed (Figure 5E) while the maximum activity for an increase in axonal length growth lies in the upper nanomolar range (Figure 5A). IGF-1 is used as a positive control for axonal outgrowth promotion (Ishii et al., 1993) and is similar efficient as NRP but less potent when used at lower concentrations (Figure 5A). When administering rat 21 mer NRP-9 during differentiation of NSC together with BrdU for 24 hrs, an increase in the proliferation rate can be observed (Figure 5H), which makes it likely, that the increased neuronal percentage in the assay is at least partly due to proliferation of neuronal progenitor cells. NRP-7SW and NRP-2KS had axonal outgrowth-promoting activity of differentiating neural precursor cells with similar efficacy as IGF-1 does but with much higher potency than

IGF-1 (Figures 5A and 5B). Quantification of these data for NRP-2KS is given as examples in Figure 5C (vehicle treatment) and in Figure 5D (NRP-2KS treatment). Over a wide concentration range NRP-2KS increased the percentage of neuronal progeny from NSC, plated on laminin in differentiation medium when normalising the neuronal cell number to the total viable cell number within the differentiation assay. At a concentration of lOnM a maximum of 2-fold increase in beta-III-tubulin-positive neurons was observed (Figure 5F). When administering NRP-2KS during differentiation of NSC together with BrdU for 24 hrs, an increase in the proliferation rate can be observed (Figure 5G), which makes it likely, that the increased neuronal percentage in the assay is at least partly due to proliferation of neuronal progenitor cells.

Example 5: Use of NRPs to Promote Neural Repopulation In Vivo In light of the findings described herein, we carry out in vivo studies to detennine whether NPRs can promote repopulation of neural tissue in animals. OEG cells have been evaluated as a source of cells for repopulation of neural tissue after injury, such in spinal cord injury. OEG cells are obtained using methods known in the art and are grown in cell culture. NPRs are added to the OEG cells in culture and/or are co-administered along with OEG cells in transplantation procedures of the spinal cord. Patients with spinal cord injury are prepared for surgery at the site of damage, the spinal canal is accessed using methods know in the art, and the area of damage identified. OEG cells and NRPs are transplanted into the site of injury and, optionally additional sources of NRPs are provided locally.

Example 6: Expression of NRPs Methods NRP Gene Expression Analysis Expression of the annotated mouse NRP gene was confirmed by RT-PCR, Northern blot and in situ hybridisation. In situ Hybridisation and Northern Blot An 88-mer oligonucleotide encompassing the mouse NRP (NRP-7 (SEQ ID NO: 35)) specific coding region upstream of the alkB homologue gene transcriptional start site was cloned between the BamHI and EcoRI sites of pGEM7Zf(-) (Promega, Madison, WI, USA). The complimentary synthetic oligonucleotides; mfsNRP.S88, sense strand is shown below. 5'AATTCGGAATGAATCGAAACCCTGGAGTCGTGACCCCGGAAGAACCTGCCAGA GCCGGAATTTCGAGTTCTGCTTCCGGGCCAAACTG SEQ ID NO: 41 and mfsNRP.AS88, the antisense strand is shown below.

5'GATCCAGTTTGGCCCGGAAGCAGAACTCGAAATTCCGGCTCTGGCAGGT TCTTCCGGGGTCACGACTCCAGGGTTTCGATTCATTCCG SEQ ID NO: 42 (Invitrogen), that also provided the appropriate 5' overhangs (underlined) were denatured and annealed at 48 μM each oligonucleotide in lOOmM NaCl, 0.1 mM EDTA (Sigma), 20mM HEPES pH7.9 by heating 2 min at 95°C, cooling to 60°C at 1°C /min and maintaining at 60°C for another 1.5 h. One μl annealed 88-mer and 300ng Qiaex extracted (Qiagen), gel purified, BamHI (Roche Diagnostics Ltd., Auckland, New Zealand) EcoRI (New England Biolabs Inc.) double digested pGEM7Zf(-) were ligated 19 h at 4°C with 0.2 unit T4 ligase (Epicentre, Madison, WI, USA). Clones prepared in competent DH10B (Invitrogen) were screened for the presence of a Clal (Roche) resistant plasmid and excision of the 88-mer upon EcoRI/BamHI double digest. Templates for synthesis of sense and anti-sense RNA probes were prepared by digesting lOμg DNA (prepared using a JetStar Maxi Kit, Genomed) to completion with 20U of either BamHI or EcoRI, respectively. The templates were gel purified using a Concert Rapid Gel Extraction System (Invitrogen). Riboprobes were transcribed and Northern blots performed using the DIG

Northern Starter Kit (Roche). 10 μg total RNA from perinatal astrocytes or pUSE- myc-mNRP-transfected HEK cells were separated on a 1.2 % formaldehyde RNA gel with 0.16-1.77 kb RNA ladder and transferred in SSC to a positively charged nylon membrane (Roche). The marker lane was cut off and stained with methylene blue. The DIG-labelled NRP probe was hybridized at 52°C over night. After stringent washes and DIG-antibody incubation, the signal was detected by CDP* luminescence with a Bioimaging System (UVP). Whole brains were extracted from El 5 mice, fixed in 4%PFA for 3 hrs, cryoprotected in 20% Sucrose o/n, embedded in Tissuetek OCT medium and stored at -80°C. The cryoprotected brains were cut into 14 μm sections on PLL coated slides, treated with 8ug/mal Proteinase K for 8min, post-fixed with 4%PFA for 5min, and hybridised o/n at 45°C with DIG -labelled NRP probe (88-mer Probe sequence) (1:100 dilution), with sense controls. After labelling with anti-DIG antibody and color development of the signal with NBT/BCIP, the sections were double labelled with nestin/GFAP/b-tubulin, and were visualised using fluorescent secondary antibody.

Results Employing PCR the NRP message was detected in different regions of the El 5 embryonic mouse brain, but interestingly the expression level was much higher in cultured mouse neural stem cells (Figure 6A). Neural stem cell specific expression was further substantiated by in situ hybridisation for mouse NRP mRNA and double staining with the stem/progenitor specific intermediate filament nestin (Lendahl et al. 1990) (Figures 6E-6H). In order to assess, whether the NRP message (mRNA) would be maintained in neuronal or glial progeny from NSC, the expression level in undifferentiated NSC was compared to NSC differentiated into astrocytes with CNTF and NSC coaxed to differentiate into neurons by BDNF. The NRP expression level was markedly increased only in the CNTF treated cells, indicating astrocytic lineages as the major source for secreted protein (Figure 6B). Comparing expression levels in several tissues from El 9 mouse, it became apparent that, except for the lung, expression in non-nervous tissues was lower, or even absent (Figure 6C). Based on the relatively high expression level in astrocytes, a northern blot on RNA from perinatal astrocyte cultures was attempted, which revealed two transcripts of approximately 0.8 and 1.2 kb size, which conespond to predicted full-length transcripts, for transcription starts -114 and -509 upstream of the translation start (Figure 6D). In situ hybridisation on sections of the E15 mouse embryonic forebrain, revealed an intense signal in the marginal zone and subplate, complemented by a lighter labelling of the cortical plate (Figure 61). Mouse NRP mRNA was co-localized with nestin-immununoreactivity. At this stage no co-labelling with GFAP or MAP2 was observed (data not shown). This staining pattern indicates expression in radial glial-like cells, undergoing transformation into immature astrocytes (Steindler & Laywell, 2003).

Example 7: Cerebellar Microexplants II - Neuroprotection Comparison of NRP-2 Segment SW (SEQ ID NO: 24 and SDF-1) Methods Cerebellar Microexplants were prepared as described in Example 9 with the addition of SDF-1 as described, in addition to glutamate/3-NP, and NRP. Results The resulting injury from treatment of unprotected cerebellar microexplants for 48hrs with 0.5mM 3-NP/glutamate was in the range from 75-92% cell death. Figure 7A shows that NRP-7SW confened highly significant neuroprotection over the concentration range from 100 fM to lOOnM and showed almost 50% recovery from injury at 100 pM. In comparison, 10 nM human SDF-1 confened less than 30% neuroprotection and revealed a nanow dose range of efficacy (Figure 7B) . The 16mer- cachexia fragment had lower efficacy and a ten-fold lower potency for neuroprotection and induction of neuronal proliferation (data not shown).

Example 8: Induction of Neuronal Migration: Comparison of NRP-9 Segment SD (SEQ ID NO: 34), NRP-2 segment SW (SEQ ID NO: 24) and SDF-1

Method for Haptotactic Migration Assay A haptotactic migration assay was performed according to the description of Example 10 using rat NRP-9SD (SEQ ID NO:34) (4nM) and mouse NRP-7SW (SEQ ID NO:24) (0.4nM) as attractants in a Boyden chamber Method for Thalamaco-Cortical Cultures The occipital cortex and dorsal thalamus from newborn Long Evans rats (PO) was dissected, according to the PO atlas of Paxinos³⁷. Occipital cortex was coronally, dorsal thalamus frontally cut with a tissue shopper (Mcllwain) into 350 μm-thick slices, which were transfened immediately into Gey's Balanced Salt Solution (GBSS) supplemented with 0.65% D-glucose (Merck) and allowed to recover at 7°C for one hour. Thalamic slices with peφendicular orientation were selected under a stereomicroscope and arranged with cortical tissue at a distance of at least 3 mm on cover slips. In this case, the thalamus was orientated with the habenula nucleus facing cortical layer VI. The slices were adhered to the cover slips in a plasma clot by 10 μl of chicken plasma (Cocalico), coagulated with 10 μl of thrombin (25 U/ml, ICN). Cover slips were placed in roller tubes (Nunc) and supplied with 0.75 semi- artificial culture medium [2/4 Basal Medium Eagle, 1/4 Hank's Balanced Salt Solution, 1/4 inactivated horse serum, 2 mM L-glutamine and 0.65% D-glucose. Cultures were maintained in a roller tube incubator at 36°C for up to 20 days in vitro (DIV) and media containing rat cachexia-related NRP was exchanged every three days. OTCs of rat embryonic forebrain were prepared as described. Briefly, pregnant rats at El 7 gestation were sacrificed, the fetuses rapidly removed. Coronal slices of the forebrain were cut at 400 μm thickness and placed onto millicell PICM ORG50 membranes (Millipore) in 3 cm Petri dishes containing 1 ml of DMEM/F12, 6.5 mg/ml glucose, 0.1 mM glutamine and 10 % FCS for 3-4 hrs. Afterwards the medium was exchanged to 1 ml Neurobasal B27, 6.5 mg/ml glucose, 0.1 mM glutamine, to which 10 μl PBS, NRP, or SDF-1 were added for the indicated concentrations and a small Dil (l,l'-dioctodecycyl-3,3,3'3- tetramethylindocarbocyanine) crystal of uniform size was placed in the medial ganglionic eminence (n = 10-15) under a stereomicroscope. After 24 hrs cultivation the total number or migrated neurons dispersed in the ganglionic eminence and cortex were counted under an inverted fluorescent microscope.

Results . NRP-9SD (SEQ ID NO:34) (4nM) and mouse NRP-7SW (SEQ ID NO:24) (0.4nM) exhibited substantial chemoattractive activity of the NRPs on mouse neural stem cells. Nearly four times as many cells as in control condition are attracted by the NRP-9SD while 10-fold less amount of NRP-7SW still attracted 2-fold more NSCs to the culture dish bottom. In comparison, lOOnM of SDF-1 coating attracted a similar number of cells, as did the 0.4nM mouse NRP-7SW (Figure 36A). Their representative moφhological phenotype is depicted in Figure 8B. In an OTC assay using sagittal embryonic brain slices that include the ganglionic eminence and the cortical anlage NRP-7SW administered to the OTC lead to a 5-fold increase in number of migrating neuronal precursor cells (Figure 8C). Human SDF-1 induces similar numbers of cells to migrate from the medial ganglionic eminence to the cortical anlage but as in the haptotactic migration assay the 63 amino acid long mature human SDF-1 is far less potent than NRP.

Example 9: Recombinant NRP Expression and Activity Assays Preparation of HEK293-Expressing mfsNRP HEK293 cells were fransfected with N-terminal and C-terminal tagged mNRP fusion protein expression constructs (pUSE-Flag-mNRP2 and pUSE-myc-mNRPll, respectively) using 25KDa polyethyleneimine (Boussif et al.; 1995). The NRP sequence used was SEQ ID

NO:35. Parallel transfections with pEGFP-Nl (Clontech) were also performed. Briefly, each construct was combined with PEI at ratios of 6, 9 and 12 to 1 PEI nitrogen to DNA phosphate at 10 ng/ul DNA in 5% glucose. HEK293 cells at 70% confluence in 2 ml growth medium (DMEM supplemented with 10% fetal calf serum) in 36 mm wells were fransfected by 4.5 h incubation with 200 ul of transfection mix per well. After this, 2 ml growth medium with 2x antibiotic/antimycotic mix were added to each well. The following day, the cultures were split

1:15 using 1 ml/well Trypsin-EDTA into growth medium containing 800 μg/ml of geneticin.

Selection was maintained for 17 days with changes of medium twice a week. Upon confluence, selection was continued on a portion of cells at a 1:10 split and the remainder were frozen in growth medium containing 10% DMSO. Additionally, a 1 :2 split from the 800 μg/ml geneticin selection was prepared day 13 Stable transfectants were harvested for detection of expressed, tagged proteins. Western Blotting Confirmed Expression of Myc-NRP in HEK293 Cells Western blotting was carried out to confirm expression of Myc-NRP in HEK293 cells. Cell lysates were solubilised under denaturing conditions and proteins were separated using SDS polyacrylamide gel electrophoresis (PAGE). Expressed recombinant mouse Myc- NRP is detected by monoclonal anti-Myc antibody.

Results The creation of full-length recombinant mouse NRP under the control of a c-myc promoter fransfected into HEK-cells revealed an expected 0.8 kb-sized transcript when probed with the antisense 88 bp NRP cRNA (Figure 9A). The 16.5kDa recombinant protein product expressed by the Myc-NRP-HEK cells migrated on a Laemmli SDS-gel to the protein marker size of 20 kDa (Figure 9B). The measured biological activities of the expressed recombinant complete NRP gene product encoded by the NRP gene equalled those of the tested NRP synthetic peptides. In a co-culture assay together with cerebellar microexplants Myc-NRP-HEK cells confened substantial neuroprotection when seeded at different cell concentrations, with a recovery value of 51% of MAP-2-positive neurons at a Myc-NRP-HEK cell number of 5000 (Figure 9C). Control Myc-HEK possessing the empty vector only, did not reveal any recovery of MAP-2-positive cells after oxidative/excitotoxic stress. The added recombinant HEK cells revealed a bell-shaped dose response curve for neuroprotection. The Myc-NRP-HEK cells displayed chemoattractive neuronal migration-inducing activity when tested in the haptotactic migration assay using mouse NSCs. When mouse NRP expressing Myc-NRP-HEK cells were seeded onto the bottom of the Boyden chamber and mouse NSCs were seeded into the inserts, the detected neuronal cell population after 24hrs displaying neurite outgrowth was two-fold increased compared to the same assay using control Myc-HEK cells (Figure 9D).

Example 10: Possible Mechanism of Action of NRPs Recent data suggest that SDF-1 (CXCL12) and its receptor CXCR4 may have parallel effects in the immune and nervous systems: they can regulate cellular movement, proliferation, plasticity (neurite outgrowth and differentiation) and survival of neurons and lymphocytes (Vlahakis et al., 2002 and Lazarini et al., 2003). Parallel reliance on CXCL12 might support coordinated homeostatic interactions but might also constitute a unique vulnerability to inflammatory processes, as HIV-1 infection and subsequent suffering of neuropathy, as a result to CXCR4 receptor binding (Keswani et al., 2003). HIV and related viruses require co-receptors, in addition to the lymphocyte receptor CD-4, to infect target cells. One of the HTV-used co-receptors is the G-protein coupled chemokine receptor CXCR4. SDF-1 interaction with CXCR4 can prevent HTV entry into the CD4-lymphocyte. Moreover, it is known that cancer metastasis can be prevented by inliibiting the migration invasion of cancer cells by antagonizing the CXCR4-receptor in animal models (Rubin et al., 2003, Liang et al, 2004). So far, SDF-1 is the only known ligand binding to the CXCR4 receptor. We provide here some evidence that the NRPs can represent a new class of ligands for the CXCR4 receptor and that biological activity (e.g. chemoattraction and neuronal survival) can be exerted by activation of CXCR4 receptors. Thus, NRPs can be agonists for the CXCR4 receptor without excluding the possibility that there might be antagonistic effects of NRPs on the CXCR4 receptor as well. Similar observations were made for SDF-1: single amino acid substitution can antagonize SDF-1 effects on CXCR4 (Tudan et al., WO0185196). It should be understood however, that this is not the only possible mechanism of action of NRPs, and that other mechanisms may account for the observations described herein. Methods Cerebellar microexplants were prepared as described in Example 9 with the addition of SDF-1, Wortmannin and PD98059 Cboth Calbiochem), as described, in addition to glutamate/3-NP, and NRP.

Effects of Neutralizing Antibodies Against CXCR4 Receptor on Survival- Promoting and Migration-Inducing Activities of NRPs Application of lug/ml of a neutralizing antibody raised against the CXCR4 receptor (fusin receptor) for 72 hours significantly decreased neuronal survival and neuronal migration patterns within cerebellar microexplants treated with NRPs. At an antibody concentration of 1 μg/ml, neuroprotective and/or migration-promoting effects of NRP-9 SD (SEQ ID NO: 34) were abolished (Figure 10A). Similar inhibition was observed for the human NRP-2KS (SEQ ID NO:23) (Figure 10B) or mouse NRP-7SW (SEQ ID NO:24) (data not shown). Effects of Neutralizing Antibodies Against CXCR4 Receptors on Chemoattractive Actions of NRPs Chemoattractive effects of lOng/ml NRP coated to the culture dish were blocked by pre-incubating the neuronal stem cell line MEB-5 for 1.5 hrs with a neutralizing antibody for CXCR4. Significantly fewer cells migrated compared to the migration observed with NRP-9 SD (SEQ ID NO:34) peptide alone (Figure 10C).

NRP Action Can Be Mediated by ERKl/2 and Akt Phosphorylation To investigate the signalling pathways utilized by NRP to exert neuroprotective and/or migratory effects we used the MEK inhibitor PD98509 and the phosphatidylinositol 3- kinase (PI-3K) inhibitor wortmannin to block MAPK or Akt phosphorylation. To address the role of MAPK and the upstream regulator of akt activity PI-3K (phosphorylation of Akt) in the mechanism of action of NRP-mediated neuroprotection and chemoattraction we tested the MAPK kinase (MEK) inhibitor PD98509 for efficacy in the cerebellar microexplant and haptotactic migration assays. The vehicle and injury treatment with and without PD98509 revealed only modest toxicity of the inhibitor, while simultaneous treatment of the inhibitor with NRP-9 SD (SEQ ID NO: 34) abolished the neuroprotective activity of NRP (Figure 11 A). The inhibition of Akt phosphorylation with wortmannin (100 nM) had no significant effect after vehicle and injury conditions without the NRP, but similar to the inhibition of ERKl/2 phosphorylation, abolished neuroprotection produced by NRP (Figure 11B). In the haptotactic migration assay the nearly three-fold increase in migrating NSC was almost completely blocked with PD98509 (from 0.1 pM to 100 pM), with no significant effect on basal migration in the BSA coated wells (Figure 11C). In contrast to neuroprotection, the chemoattractive migratory activity of NRPs was not suppressed by inhibition of PI-3K with wortmannin (data not shown). To exclude that the reduced number of migrated cells at the bottom compartment could have been due to impaired neuronal survival, the insert was stained for live cells with Syto21 and the upper surface analysed. No significant difference in the number of surviving cells was found.

Example 11: NRP Efficacy in Vivo Materials and Methods To test the efficacy of NRPs in vivo studies were carried out in rats that had been exposed to hypoxic-ischemic injury (HI). Adult rats (50 days old, Wistar, 250-300g, male) were used. The modified Levine model preparation and experimental procedures were used (Rice et al, 1981, Ann. Neurol. 9: 131-141; Guan et al J., 1993, Cereb. Blood Flow Metab.: 13(4): 609-16). These procedures in brief, consist of an HI injury induced by unilateral carotid artery ligation followed by inhalational asphyxia in the animals with an implanted lateral ventricular cannula. A guide cannula was stereotaxically placed on the top of the dura 1.5mm to the right of the mid-line and 7.5mm anterior to the interaural zero plane under halothane anaesthesia. The right carotid artery was double ligated two days after the cannulation. After a 1-hour recovery period from anaesthesia, each of the rats was placed in an incubator where the humidity (90±5%) and temperature (31^o±0.5°C) were controlled for another hour, and then each of the rats was exposed to hypoxia (6% oxygen) for 10 min. The animals were kept in the incubator for an additional 2 hours before treatment. Rats were treated intracerebral ventricularly (icv) with 5nM (n=12), 50nM (n=12) or 500nM of NRP-4 segment PQ (PGRAEAGGQ; SEQ ID NO:43) dissolved in saline, or vehicle (n=10) (normal saline) 2 hours after hypoxic-ischemic insult. Histological examination was perfonned on rats 5 days after the hypoxic-ischemic injury. The rats were killed with an overdose of sodium pentobarbital and were perfused transcardially with normal saline followed by 10% formalin. The brains were kept in the same fixative for a minimum of 2 days before being processed using a standard paraffin imbedding procedure. Coronal sections 8 μm in thickness were cut from the striatum, cerebral cortex and hippocampus and were stained with thionin and acid fuchsin. The severity of tissue damage was scored in the striatum, cortex and the CA1-2, CA3, CA4 and dentate gyms of the hippocampus. Tissue damage was identified as neuronal loss (acidophilic (red) cytoplasm and contracted nuclei), pan-necrosis and cellular reactions. Tissue damage was scored using the following scoring system: 0: tissue showed no tissue damage, 1: <5% tissue was damaged, 2: <50% tissue was damaged, 3: >50% tissue was damaged and 4: >95% tissue was damaged. Results and Conclusion The results of this study are shown in Figure 12. The 9mer-fragment of human chromosome 15, NRP-4 segment PQ (PGRAEAGGQ-NH₂; SEQ ID NO:43; 9 ng/20μl icv 2 hrs after hypoxia), confened 100% neuroprotection within all analysed brain regions five days after insult.

Example 12: Growth Promotion of Olfactory Ensheating Glial ("OEG") Cells To determine whether NRPs might be useful for remyelinating neural tissues, we carried out studies using human olfactory ensheating glial (OEG) cells. OEG cells are being evaluated as a source population of cells for repopulating damaged neural tissue, such as that of the spinal cord. OEG cells were obtained from scrapings of human nasal mucosa using methods that are known in the art. Cells were grown in medium under control conditions and in the presence of NRP-7 SW (SEQ ID NO:24). Under control conditions, the normal rate of proliferation of OEG cells is about 25% within 72 hours. We found that in the presence of NRP-7 SW (SEQ ID NO:24), the OEG cells grew at a rate of about 50% over 72 hours. These results indicate that NRPs can enhance proliferation of OEG cells, and thereby can be useful in neural cell transplant procedures to promote cell growth and return of neural function to damaged tissues (Figure 13)

Example 13: Neuroprotective Activity of NRP-5 Segment RG Peptide Analogues To determine if alteration in the amino acid sequence of peptides related to NRP-5 Segment RG produced peptides having activity different from that of NRP-5 Segment RG (SEQ ID NO:30), we produced synthetic peptides having various amino acid substations. We tested substituted NRP-5 RG peptides in the cerebellar microexplant assays described herein. Amino acid substitutions within the first N-terminal five amino acids of the amidated peptide having amino acid sequence REGRRDAPGRAGG-NH₂ (SEQ ED NO:30) produced a peptide having the sequence: REAAADAPGRAGG-NH₂ (SEQ ID NO:44) and AAARRDAPGRAGG-NH₂ (SEQ ID NO:45). Amino acid substitutions in the PGR-domain resulting in a peptide having the amino acid sequence REGRRDAAAAAGG-NH₂ (SEQ ID NO:47) and substitution of the C- terminal GG-domain resulting in a peptide having the amino acid sequence REGRRDAPGRAAA-NH₂ (SEQ ID NO:46). Each of the above substituted peptides were tested and the results compared to those obtained using NRP-5 Segment RG (SEQ ID NO: 30; "NRP-5 RG"). Figure 14A shows that SEQ ED NO: 44 had neuroprotective activity with a maximum effect observed at a concentration of about 1 pM. The bars on the right, labeled "standardl3mer," refer to NRP-5 RG (SEQ ID NO:30). The substitution of amino acids 3-5 of NRP-5 RG did not significantly change in the activity profile of the peptide (Figure 42A). Figure 14B shows that SEQ ID NO:45 had neuroprotective activity with a maximal effect observed at a concentration of about 1 pM. The bars on the right, labeled "original," refer to NRP-5 RG (SEQ ID NO:30). Substitutions in positions 1-3 producing AAARRDAPGRAGG-NH₂ (SEQ ED NO:45) resulted in even higher neuroprotective activity than the original NRP-5 RG. The result may be due to the higher stability profile of the analogue over NRP-5 RG, although other hypotheses may account for the observation. Figure 42C shows that SEQ ID NO:46 had less effect than NRP-5 RP (SEQ ED NO:30; right bars; "standardl3mer") or of SEQ ID NO:44 or SEQ ID NO:45 (Figures 14A and 14B, respectively). In fact, the only statistically significant effect was observed at a concentration of 100 nM. The original sequence (SEQ ID NO:30) produced 20% higher neuroprotection with IO⁶ to IO⁷ times higher potency than SEQ ID NO:46. Figure 14D shows that SEQ ID NO:47 had some neuroprotective effect at certain concentrations (e.g., 0.1 pM, 10 pM and 100 nM), but at other concentrations, had no effect compared to those observed for NRP-5 RG (SEQ ID NO:30; right bars labeled "standard"). We conclude from these studies that both the PGR and the C-terminal GG domains are useful for maintaining the activity of NRP-5 RG (SEQ ID NO:30). We also conclude that the amidated C-tenninus of an NRP is not sufficient to produce neuroprotective effects, because the C-terminal amidated NRP, SEQ ID NO:46, produced little if any neuroprotective effect at most concentrations. Further, alteration in the interior of an NRP did affect activity, even though none of the interior amino acids has a C-terminal amide group.

Example 14: Proliferation-Inducing and Chemoattractive Activities of NRP-5 RG In another set of studies, we determined effects of NRP-5 RG on induction of neural cell proliferation in embryonic cerebellar microexplants as described herein. Figure 15 shows that NRP-5 RG exhibited proliferation-inducing activity with a maximal activity observed at a concentration of 100 pM. Some effect was observed at concentrations of 1 mP and even at 0.1 pM, but those effects were not statistically significant. In additional studies, we studied chemoattractive effects of NRP-5 RG in a haptotactic migration assay as described herein. Figure 16 shows that NRP-5 RG was chemoattractive, and had 42.1% greater effect than cells exposed to control (BSA-containing) medium.

Example 15: NRPs Protected Neural Cells In Response to Oxidative Stress In this series of experiments, we studied whether NRP-4 Segment PQ ("NRP-4 PQ") was able to protect cerebellar microexplanted cells from oxidative injury. We exposed cells to vehicle, 0.1 mM hydrogen peroxide or different concentrations of NRP-4 PQ in vehicle. Figure 17 shows neuroprotective effects of NRP-4 PQ after 48hrs of oxidative stress in response to O.lmM hydrogen peroxide. The neuroprotection associated with NRP-4 PQ treatment was even greater than that of control explants receiving no peroxide. Although the mechanism for this potentiation of neurite outgrowth is uncertain, it may relieve the stresses associated with creating or maintaining the microexplants themselves. These findings are relevant to many conditions in which oxidative stress plays a role. For example, oxidative stress is associated with all both and chronic CNS injuries and diseases. Inhibition of oxidative-stress mediated neurotoxicity through NRP action can be highly beneficial for many CNS injuries or diseases.

Example 16: Neuroprotection Mediated by Phosphorylated NRP-7 SW We earned out a series of studies to determine whether phosphorylation of the N- terminal amino acid serine altered neuroprotective effects of NRP-7 Segment SW ("NRP-7 SW"). Phosphorylation of NRP-7 SW produces NRP-7 ^PSW. We hypothesized a role for N- terminal serine phosphorylation because of the high likelihood prediction (0.9) that serine is phosphorylated under in vivo conditions (NetPhos 2.0 Server - Technical University of Denmark). Cerebellar microexplants were injured with 0.5mM 3-NP/glutamate and treated with NRP-7 ^PSW. Figure 18 shows results of experiments that demonstrate that NRP-7 ^PSW exhibited neuroprotective effects, with significant effects observed at concentrations as low as 0.1 pM, which exhibited more neuroprotective activity (44.2% neuroprotection) than other concentrations tested. Because the lowest concentration used (0.1 pM) exhibited substantial neuroprotective effects, lower concentrations will also exhibit neuroprotective effects.

Example 17: Enhancement of Proliferation of NSCs After Induction of Differentiation by NRP-9 SD El 5 mouse NSC P10 cells were plated on laminin and cultured for 3 days in the presence of NRP-9 SD (SEQ ID NO:34) and BrDU for the last 48 hours of the culture period. For each condition 4 visual fields in two independent wells were counted and the number of proliferating cells were determined. Results NRP-9 SD increased the rate of proliferation of differentiating mouse NSC precursor cells (Figure 19). This biological activity was observed over a wide dose range from 100 fM to 1 nM. Example 18: Expression of NRPs II In Situ Hybridisation Whole brains were extracted from El 5 and El 7 mice, fixed in 4% paraformaldehyde (PFA) for 3 hrs, cryoprotected in 20% sucrose overnight (o/n), embedded in Tissuetek OCT (Sakura finetek) and stored at -80°C. The cryoprotected brains were cut into 14 μm thick sections, placed on PLL coated slides, treated with 8ug/mal Proteinase K for 8min, post-fixed with 4% PFA for 5 min, and hybridised overnight at 45°C with DIG-labelled NRP sense and antisense probes (88-mer Probe sequence described in Example 13; sense strand: SEQ ID NO:41 and antisense strand: SEQ ID NO:42; 1:100 dilution). After labelling with anti-DIG antibody and color development of the signal with NBT/BCIP, the sections were double labelled with nestin/GFAP/βlll-tubulin and vimentin 1:100 dilution (abeam, mouse monoclonal [RV202], ab8978-l), and were visualised using fluorescent secondary antibody. The sense controls remained negative.

Results Figures 20A and 20B depict fluorescence micrographs of brains of animals as described above. The co-localization of the mouse frameshift NRP message with the radial glia marker, vimentin, indicated that NRP expression was maintained in the neuroepithelial stem cell - radial glia -astrocytic lineage (Figures 20 A and 20B). This points to an important function in neural stem cells, as radial glia have been shown to generate neurons in many brain regions during development (Anthony TE, Klein C, Fishell G, Heinz N. 2004. Radial glial cells serve as neuronal progenitors in all regions of the central nervous system. Neuron 41:881-890) and astrocytes of the adult subventricular zone have been demonstrated to be neural stem cells (Doetsch F, Caile I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A. 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703-706). We conclude from these studies that NRP mRNA is produced in the brains of mice.

Example 19: Re-Sequenced SEQ ID NO:8 and SEQ ID NO:9 On August 20, 2004, a report of revised sequences conesponding to SEQ ID NO:8 and SEQ ID NO:9 were published (NBCI, NT 026446). Based on the newly identified sequences, we have annotated a nucleotide NRP sequence consisting of 225 nucleotides coding for a peptide having 75 amino acids. According to the previously annotated sequence on chromosome 15 coding for NRP (SEQ ID NO:8), the following has changed within the sequence: position 168 has changed from a G to a C; otherwise exons 1-4 remained unchanged (including the active biologically sequence for NRP). From position 179 onwards the newly annotated exon 5 starts. From position 222 onwards exon 6 starts.

5'- 9 18 27 36

ATG GCT GTT GTG TTA CTT GCA CCA TTT GGG GAC ATC AGC CAG Met Ala Val Val Leu Leu Ala Pro Phe Gly Asp lie Ser Gin 45 54 63 72 81 GAA ATC ACA AAG GTT GGG ACA GGG ACT CCA GGG AGG GCT GAG Glu lie Thr Lys Val Gly Thr Gly Thr Pro Gly Arg Ala Glu 90 99 108 117 126

GCC GGG GGC CAG GTG TCT CCA TGC CTG GCG GCG TCC TGC AGT Ala Gly Gly Gin Val Ser Pro Cys Leu Ala Ala Ser Cys Ser 135 144 153 162

CAG GCC TAT GGC GCC ATC TTG GCT CAC TGC AAC CTC TGC CTC

Gin Ala Tyr Gly Ala lie Leu Ala His Cys Asn Leu Cys Leu

171 180 189 198 207

CCA GGT TCA AGT GAT CTG CCT GCC TCA GCC TCC CAA AGT GCT

Pro Gly Ser Ser Asp Leu Pro Ala Ser Ala Ser Gin Ser Ala 216 225 228 (STOP)

AGG TTA CAG GTT GAT TAA SEQ ID NO:48

Arg Leu Gin Val Asp # SEQ ID NO:49

The whole amino acid sequence written in the one letter code:

>humchroml5NRPexonl 2 3 4 5 6 MAWLLAPFGDISQEITKVGTGTPGRAEAGGQVSPCLAASCSQAYGAILAHCNLCLP GSSDLPASASQSARLQVD SEQ ID NO:49

Example 20: Treatment of Stroke A patient presents with symptoms of stroke. A diagnosis of stroke is made and the physician then administers a NRP compound to the patient intravenously or alternatively, directly into the cerebral ventricle or directly into the affected portion of the patient's brain.

The NRP compound is a peptide or protein as described herein and is administered in a phannaceutically acceptable form, including, if desired, excipients, buffers and stabilizers.

Treatment with the NRP decreases the neurodegeneration associated with the stroke and an expected worsening of symptoms at least partially slows, stops or is at least partially reversed. Example 21 : Prophylactic Use of NRPs A patient is diagnosed with a cardiac vascular insufficiency and coronary artery bypass (CABG) surgery is indicated. CABG surgery is associated with reduced cerebral perfusion, which can lead to hypoxic or ischemic brain injury. To decrease adverse effects of such hypoxia or ischemia, the patient is pre-treated with a NRP compound. The NRP compound is administered to the patient in a pharmaceutically acceptable form, including, if desired, excipients and/or stabilizers. Routes of administration include intravenous, intercerebrally, or via a cerebral ventricle. If desired, multiple routes of administration can be used. Pre-treatment of a patient undergoing CABG surgery decreases the neurodegeneration associated with CABG surgery and the patient experiences reduced post-surgical neurological deficits compared to patients undergoing CABG surgery without pre-treatment with a NRP.

This invention is described with respect to specific embodiments thereof. Those of ordinary skill in the art without undue experimentation may develop other embodiments incoφorating the disclosures and teachings of this application. All of these embodiments are considered to be part of this invention. All references cited herein are incoφorated fully by reference.

REFERENCES Akerblom, IE, and Murry, LE (1996). Human cachexia associated protein. US patent 5,834,192. Anderson, CV, Wood, DM, Bigler, ED, and Blatter, DD (1996). Lesion volume, injury severity, and thalamic integrity following head injury. J. Neurotrauma 13: 35-40. Anderson, WF (1992) Human gene therapy. Science 256: 808-813. Bach et al., (1995) Insulin like growth factor binding proteins. Diabetes Reviews 3: 38-61. Baldwin, ME, Roufail, S, Halford, MM, Alitalo, K, Stacker, SA and Achen, MG (2001). Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis. JBiol Chem 276: 44307-44314. Beal, MF, Kowall, NW, Ellison, DW, Mazurek, MF, Swartz, KJ, and Martin, JB (1986). Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid. Nature 321: 168-171. Bolz, J, Novak, N, and Staiger, V (1992). Formation of specific afferent connections on organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus. J. Neurosci. i2:3054-3070. Brose, K, and Tessier-Lavigne, M (2000). Slit proteins: key regulators of axon guidance, axonal branching, and cell migration. Curr. Opin. Neurobiol. 10: 95-102. Cote, F, Do, TH, Laflamme, L, Gallo, J-M, and Gallo-Payet, N (1999). Activation of the AT2 receptor of angiotensin II induces neurite outgrowth and cell migration in microexplant cultures of the cerebellum. J. Biol. Chem. 274: 31686-31692. Cunningham, TJ, Hodge, L, Speicher, D, Reim, D, Tyler-Polsz, C, Levitt, P, Eagleson, K, Kennedy, S, and Wang, Y (1998). Identification of a survival promoting peptide in medium conditioned byoxidatively stressed cell lined of nervous system origin. J. Neurosci. 18: 7047-7060. De Curtis, I, and Reichardt, LF (1993). Function and spatial distribution in developing chick retina of the laminin receptor α6βl and its isoforms. Development 118: 377- 388. Dodd, J, Morton, SB, Karagogeos, D, Yamamoto, M, and Jessell, TM (1988). Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons. Neuron 1: 105-116. Dyke, MW, Bianchi-Scana, G, and Musso, M (2001). Characterization of a triplex DNA-binding protein encoded by an alternative reading frame of loricrin. Eur. J. Biochem 268: 225-234. Fallon, J, Reid, S, Kinyamu, R, Opole, I, Opole, R, Baratta, J, Korc, M, Endo, TL, Duong, A, Nguyen, G, Karkehabadhi, M, Twardzik, D, and Loughlin, S (2000). In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. PNAS 97: 14686-1491. Fe i, RT, and Levitt, P (1995). Regulation of regional differences in the differentiation of cerebral cortical neurons by EGF family-matrix interactions. Development 121: 1151-1160. Fueshko, S, and Wray S (1994). LHRH cells migrate on peripherin fibers in embryonic olfactory explant cultures: an in vitro model for neurophilic neuronal migration. Dev. Biol 166: 331-348. Gahwiler, BH (1981). Organotypic monolayer cultures of nervous tissue. J. Neurosci. Methods 4: 329-342. Ganzler, SI, and Redies, C (1995). R-cadherin expression during nucleus formation in chicken forebrain. J. Neurosci. 15: 57-72. Gomez, TM, and Spitzer, NC (1999). In vivo regulation of axon extension and pathfindingby growth-cone calcium transients. Nature 397: 350-355. Gulyas, Al, Hajos, N, and Freund, TF (1996). Interneurons containing calretinin are specialized to control other neurons in the rat hippocampus. J. Neurosci. 16: 3397-3411. Guth, S, Tange, TO, Kellenberger, E, Valcarcel, J (2001). Dual function for U2AF35 in AG-dependentpre-mRNA splicing. Mol Cell Biol ^' 21: 7673-7681. Hatten, ME, and Heintz, N (1999). Neurogenesis and migration - In Fundamental Neuroscience, (R: Zigmond, ed.), pp 451-479, Academic Press, San Diego. Hermann, DM, Mies, G, Hata, R, and Hossmann, KA (2000). Microglial and astrocytic reactions prior to onset of thalamic cell death after traumatic lesion of the rat sensorimotor cortex. Ada Neuropathol (Berl) 99: 147-153. Hughes, PE, Alexi, T, Williams, CE, Clark, RG, and Gluckman, PD (1999). Administration of recombinant human Activin-A has powerful neuiOtrophic effects on select striatal phenotypes in the quinolinic acid lesion model of Huntington's disease. Neuroscience 92: 197-209. Hwang et al., (1980). Hepatic Uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study. Proc. of the Natl. Acad. Of Sciences USA 77: 4030-4034. Ishii, N, Wadsworth, WG, Stem, BD, Culotti, JG, and Hedgecock, EM (1992). UNC-

6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9: 873-881. Langer et al., (1981) Biocompatibility of polymeric delivery systems for macromolecules. J. Biomed. Mater. Res. 15: 27-277. Liang, S, and Cratcher, KA (1992). Neuronal migration in vitro. Dev. Brain Res. 66:

127-132. Lu, Q., Sun, E., Klein, R. S. and Flanagan I. G.(2001). Ephrin-β reverse signalling is mediated by a novel PDZ-RGS protein and selectively inhibits G-protein coupled in chemoattraction. Cell 105: 69-79. Nakao, N, and Itakura, T (2000). Fetal tissue transplants in animal models of

Huntington's disease: the effects on damaged neuronal circuitry and behavioural deficits. Prog. Neurobiol. 61: 313-338. Obst, K, and Wahle, P (1995). Areal differences of NPY mRNA expressing neurons are established in the late postnatal rat visual cortex in vivo, but not in organotypic cultures. Eur. J. Neurosci. 7: 2139-2158. Pasterkamp, RJ, Giger, RJ, Baker, RE, Hermens, WT, and Verhaagen J (2000). Ectopic adenoviral vector-directed expression of Sema3 A in organotypic spinal cord explants inhibits growth of primary sensory afferents. Dev. Biol. 220: 129-141. Paxinos, G, Toerk, I, Tecott, LH, and Valentino, KL (1991). Atlas of the Developing Brain. Academic Press: San Diego. Polleux, F, Monow, T, and Ghosh, A (2000). Semaphorin3A is a chemoattractant for cortical dendrites. Nature 404: 567-573. Rozas, G, Liste, I, Lopez-Martin, E, Guena, Mj, Kokaia, M, and Labandeira-Garcia, JL (1996). Intrathalamic implants of GABA-releasing polymer matrices reduce motor impairments in rats with excitotoxically lesioned striata. Exp. Neurol. 142: 323-330. Sieg, F, Obst, K, Gorba, T, Riederer, B. Pape, H-C, and Wahle, P (1998). Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo. Dev. Brain Res. 110: 83-95. Stoppini, L, Buchs, P-A, and Muller, D (1991). A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37: 173-182. Van der Flier, A, Kuikman, I, Kramer, D, Geerts, D, Kreft, M, Takafuta, T, Shapiro, SS and Sonnenberg, A (2002). Different splice variants of filamin-B affect myogenesis, subcellular distribution, and determine binding to integrin β subunits. J Cell Biol 156: 361- 376. Wagener, R, Kobbe, B, Aszodi, A, Aeschlimann, D, and Paulsson M (2001). Characterization of the mouse matrilin-4 gene: A 5' antiparallel overlap with the gene encoding the transcription factor RBP-L. Genomics 16: 89-98. Wagner et al., (1990) Transfenin-polycation conjugates as earners for DNA uptake into cells. Proc. Natl. Acad. Sci. USA. 87: 3410-3414. Wu et al., (1987) Receptor-mediated in vitro gene transformation by a soluble DNA canier system. J. Biol. Chem. 262: 4429-4432. Yamamoto M, Hassinger, L, and Crandall, JE (1990). Ultrastructural localization of stage-specific neurite-associated proteins in the developing rat cerebral and cerebellar cortices. /. Neurocytol 19: 619-627. Zhu, Y, Yu, T, Zhang, X-C, Nagasawa, T, Wu, JY, Rao, Y (2002). Role of the chemokine SDF-1 as the meningeal attractant for embryonic cerebellar neurons. Nature Neurosci. 5: 719-720.

INDUSTRIAL APPLICABILITY Embodiments of this invention include genes and peptides for neural regeneration paptides (NRPs) that are useful for manufacturing compositions for therapeutic use to treat conditions involving neurodegeneration or neural cell death occur. Treatment with NRPs can lead to increased neuronal survival, neuronal migration, neuronal differentiation, neurite outgrowth and/or neuronal proliferation. Conditions such as Alzheimer's disease, Parkinson's disease, hypoxia, ischemia, stroke and coronary artery bypass surgery can be usefully treated by compositions and methods of this invention.

Claims

We Claim:

1. A neural regeneration peptide (NRP), having an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ED NO:34, SEQ ID NO:36, SEQ ID NO:44 and SEQ ID NO:45.

2. An oligonucleotide sequence encoding an NRP, said oligonucleotide selected from the group consisting of SEQ ID NO:27, SEQ ID NO:35 and SEQ ID NO:48.

3. An oligonucleotide encoding an NRP, wherein said oligonucleotide sequence differs from the oligonucleotide sequence of claim 2 only by virtue of redundancy in the genetic code.

4. A method for promoting neural regeneration, comprising the step of delivering to a neural tissue, a pharmacologically effective amount of at least one NRP of claim 1.

5. The method of claim 4, wherein said neural regeneration is selected from the group consisting of neural survival, neural proliferation, neural migration, neurite outgrowth and neural differentiation.

6. The method of claim 4, wherein said NRP is delivered intravenously.

7. The method of claim 4, wherein said NRP is encoded by an oligonucleotide of claim 2.

8. The method of claim 4, wherein said NRP is encoded by an oligonuclotide of claim 2 or an oligonuclotide having a sequence that differs from that of an oligonuclotide of claim 2 only by the redundancy of the genetic code.

9. An oligonucleotide encoding an NRP, said oligonuclotide having at least 85% sequence identity over the entire sequence to an oligonuclotide sequence of claim 2.

10. An expression vector comprising an oligonuclotide sequence of claim 2, wherein said expression vector can express an NRP.

11. The expression vector of claim 10, wherein the vector is a plasmid.

12. The expression vector of claim 10, wherein the vector is a viral vector.

13. A host cell containing the expression vector of claim 10.

14. The host cell of claim 13, wherein the cell is prokaryotic.

15. The host cell of claim 13, wherein the cell is eukaryotic.

16. An oligonucleotide sequence of claim 2, wherein "t" is replaced by "u."

17. An oligonucleotide complementary to an oligonucleotide selected from the group consisting of SEQ ID NO:27, SEQ ID NO:35 and SEQ ID NO:48.

18. The nucleotide sequence of claim 17, said sequence at least 15 base pairs in length, and hybridizes under stringent hybridization conditions to a genomic DNA encoding an NRP.

19. A polypeptide comprising an amino acid sequence having at least 80% amino acid sequence identity over the entire sequence to an amino acid sequence selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ED NO:31, SEQ ID NO:32, SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:36, SEQ ID NO:43 SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:49.

20. The method of claim 4, wherein said neural tissue is present in an animal's basal ganglia.

21. The method of claim 20, wherein said neural tissue is at risk for degeneration due to Parkinson's Disease, Alzheimer's disease, hypoxia, ischemia, stroke, coronary artery bypass surgery, trauma or Huntington's Disease.

22. A method for treating a neurological condition characterized by neural degeneration in an organism, comprising the step of administering to affected nerves of said organism, a pharmacologically effective amount of an NRP of claim 1.

23. The method of claim 22, wherein said neurological condition is selected from the group consisting of Parkinson's Disease, Alzheimer's Disease, hypoxia, ischemia, stroke, coronary artery bypass surgery, trauma and Huntington's Disease.

24. A method for inducing at least one of neuronal proliferation and neuronal migration in a neuron, comprising administering an effective amount of an NRP of claim 1 to said neuron.

25. A method for promoting neurite outgrowth in a neuron, comprising administering an effective amount of an NRP of claim 1 to said neuron.

26. A method for promoting neuronal survival, comprising administering an effective amount of an NRP of claim 1 to said neuron.

27. The method of any of claims 24-26, wherein said neuron is present within a living animal.

28. The method of any of claims 24-26, wherein said neuron is not within the body of said animal.

29. The method of any of claims 24-26, wherein said neuron is present within the central nervous system of said animal.

30. A method of treating a patient having a spinal cord injury comprising administering an effective amount of an NRP of claim 1 to said patient.

31. A NRP of claim 1 , wherein the amino acid sequence differs from the amino acid sequence of SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:36, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 or SEQ ID NO:49 by virtue of at least one conservative amino acid substitution.

32. A method for promoting stem cell differentiation into a neuronal phenotype, comprising administering to a subject suffering from a neuronal disorder a pharmaceutically effective amount of a neural regeneration peptide (NRP).

33. The method of claim 32, wherein said NRP is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID

NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 and SEQ ID NO:49.

34. A method for promoting axonal outgrowth in a stem cell, comprising administering to a subj ect suffering from a neuronal disorder a pharmaceutically effective amount of a neural regeneration peptide (NRP).

35. The metliod of claim 34, wherein said NRP is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO.17, SEQ ID N0.18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID

NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:45.

36. A method for promoting neuroblast proliferation, comprising administering a phannaceutically effective amount of a neural proliferation peptide (NRP).

37. The method of claim 36, wherein said NRP is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID

NO: 13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:30, SEQ ED NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:49.

38. A method for inducing migration of stem cells, comprising administering a pharmaceutically effective amount of a neural regeneration peptide (NRP) to said stem cells.

39. The method of claim 38, wherein said NRP is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:l 1, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ED NO:26, SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID

NO:33, SEQ JJD NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ED NO:45, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:49.

40. A method for treating a neurodegenerative condition in a subject in need of treatment, comprising administering to said subject, a phannaceutically effective amount of a neural regeneration peptide selected from the group consisting of SEQ ID NO:28, SEQ ID NO:20, SEQ ED NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID

NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ED NO:44, SEQ ID NO:45, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:49.

41. A method for repopulating a portion of the nervous system of a mammal, comprising implanting a population of neuroblast cells in combination with a phannaceutically effective amount of an NRP, so that said neuroblast cells proliferate, differentiate and/or fonn neurites.

42. A method for decreasing neurodegeneration associated with a medical procedure, comprising administering to a subject prior to said medical procedure, a pharmaceutically effective amount of a neural regeneration peptide (NRP).

43. The method of claim 42, wherein said medical procedure is selected from the group consisting of surgery, chemotherapy and radiation therapy.

44. The method of claim 42, wherein said medical procedure is coronary artery bypass surgery.

45. The metliod of claim 40, wherein said condition is selected from the group consisting of an infection, a cerebrovascular disease, craniocerebral trauma, a demyelinating disease, a dementia, a metabolic disorder, nutritional deficiency, alcoholism, peripheral neuropathy, a condition associated with drug-induced neural damage and chemical- induced neural damage.

46. A method for treating neurodegeneration associated with neural hypoxia or neural ischemia, comprising administering to a patient having hypoxia or ischemia, a pharmaceutically effective amount of a neural regeneration peptide selected from the group consisting of SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:49.

47. The method of claim 46, wherein said hypoxia or ischemia is associated with cardiac insufficiency or stroke.

48. The method of claim 46, wherein said hypoxia or ischemia is associated with traumatic brain injury.

49. A pharmaceutical composition comprising at least one neural regeneration peptide selected from the group consisting of SEQ ID NO:28, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:45, SEQ ID NO:46 and SEQ ID

NO:49.and a pharmaceutically acceptable medium.

50. The method of claim 46, wherein said neurodegeneration is associated with bacterial, fungal or viral infection.

51. A neural regereration peptide substantially as described herein.

52. A method for using a neural regeneration peptide substantially as described herein.