JP5026083B2 - Nerve regeneration peptides and their use in the treatment of brain injury - Google Patents

Description

Related applications:
This application is US Provisional Patent Application No. 60 / 516,018 (filed Oct. 31, 2003, title of invention: “Neural Regeneration Peptides and Methods for Their Use”); Sieg & Thorsten Gorba (Attorney Docket No: NRNZ 01023 US3 DBB), US provisional patent application 60 / 585,041 (filed on July 2, 2004, title of invention: neuroregenerative peptide: a new type of chemotaxis and neurons Survival-promoting peptide (“Neural Regeneration Peptides: A New Class of Chemoattractive and Neuronal Survival Promoting Peptides”); Inventor: Thorsten Gorba & Frank Sieg (Attorney Docket No: NRNZ 01023 US4 DBB)) (Filed October 5, 2004, title of invention: neuroregenerative peptides and methods for their use in treatment of brain damage); inventor: Frank Sieg, Paul Edmond Hughe s & Thorsten Gorba (Attorney Docket No: NRNZ 01023 US5 DBB). All of the above applications are incorporated herein by reference.
Sequence list:
This application includes a sequence list submitted as a disc containing (1) a printed version of the sequence list and (2) a computer readable sequence list. This sequence listing is included herein by reference.

  The present invention is directed to compositions of oligonucleotides and peptides that promote neuronal migration, proliferation, survival, differentiation and / or neurite outgrowth and methods for their use. More specifically, the present invention is directed to the use of such peptides in the treatment of brain damage and neurodegenerative diseases. The present invention also includes novel methods for detecting neuronal cell proliferation, migration, neurite outgrowth, survival and / or differentiation.

Mild to severe traumatic brain injury (TBI) and focal or global ischemia can result in significant neuronal cell loss and brain function loss within a short time after injury. To date, no treatment is available to prevent cell death that occurs in the brain as a result of head injury or injury caused by the disease. Furthermore, to date, there are no treatments available to restore neuronal function. Symptomatic treatment is the only treatment currently available for chronic neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and multiple sclerosis. There are currently no drugs available to intervene in the disease process or prevent cell death.
It is well known that cortical-subcortical non-thalamic lesions can undergo apoptosis within the thalamus 3-7 days after injury. Retrograde thalamic degeneration is accompanied by activation of astroglia and microglia in the thalamus (Hermann et al., 2000). Non-invasive techniques such as MRI have shown that thalamic volume is small and ventricular-to-brain ratio values increase in TBI patients with non-thalamic structural lesions (Anderson et al., 1996). These findings indicate that thalamocortical excitatory projection neurons are very vulnerable to retrograde neuronal cell death, thus demonstrating the need for rescue techniques for damaged or traumatic thalamic neurons .

The function of the inhibitory neuron circuit inside the thalamus is as essential for intra-thalamic down-regulation of neuronal activity inside the thalamus as it is inside the striatum. Animals with striatal lesions similar to those resulting from Huntington's disease have been shown to have improved behavioral outcomes when implanted with a GABA-releasing polymer matrix in the thalamus (Rozas et al., 1996) . At the cellular level within the striatum, it has been shown that calbindin immune response (“calbindin-ir”) inhibitory neurons can be rescued by administration of activin A (Hughes at al., 1999).
To date, only transplants that require fetal striatum transplantation have resulted in improved or restored motor function in animal models of Huntington's disease (Nakao and Itakura, 2000). Recovery of the GABAergic system of the thalamus and striatum damaged by Huntington's disease can improve behavioral outcomes (Beal et al., 1986).
A characteristic of nervous system development is that precursor cells migrate extensively to their correct three-dimensional spatial arrangement. Such migration promotes a series of phenotypic differentiation and placement of immature neurons into the vertebral brain. In order to accurately connect approximately 10 trillion neurons, it is necessary to construct cellular mechanisms such as the formation of layered structures in higher cortical areas (see Hatten and Heintz for a review).

The cell side that correlates with the direction of movement of migrating neurons may be the frequency and magnitude of transient Ca ²⁺ changes within a single migrating cell (Gomez and Spitzer, 1999), except for the membrane The trigger for initiation and / or execution of neuronal cell migration by binding or diffusible molecules cannot be excluded.
However, many of the instructions necessary for neurite outgrowth and neuronal migration have been identified. Plasma membrane molecules belonging to the integrin receptor family interact with extracellular matrix ligands such as laminin to initiate neuronal adhesion to the lower layer (Liang and Crutcher, 1992; De Curtis and Reichardt, 1993). Control of integrin expression affects a wide range of developmental and cellular processes, including regulation of gene expression, cell adhesion, axis creation length and cell migration. Other ligands that promote cell migration include cell adhesion molecules (ie N-CAM; cadherin; TAG-1), laminin-like molecule netrin-1, neuron-glia 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 (semaphorin 3A) was discovered. Collapsin-1 has chemical repulsive activity in axon guidance and migration patterns on primary sensory neurons (Pasterkamp et al., 2000). In contrast, collapsin-1 functions as a chemoattractant to induce cortical apical processes in the neocortical area (Polleux et al., 2000). Similar chemical repulsion to axon guidance was shown by slit-1 (diffusible protein) (Brose et al., 2000).

So far, the chain events leading to the onset of neuronal migration, i.e., neuronal attachment to the subsequent onset of migration, the process of long-range migration (including turning and migration stop signals) have not yet been described.
A midbrain lesion administered simultaneously with TGF-α causes large-scale proliferation of pluripotent stem cells originating from the subventricular zone (“SVZ”) and subsequent migration of these stem cells to the striatum. (Fallon et al., 2000). However, in order to prevent long-range migration of neuronal precursors originating from SVZ, it is desirable to activate the proliferation and migration of neurons that are very close to the lesion site.
There is only one report that deals with the stromal factor chemokine (SDF-1) as a neuron migration chemoattractant. The fetal expression form of SDF-1 attracts cerebellar granule cells to migrate from the outer germ layer to the inner granule layer (Zhu et al., 2002). Nevertheless, this chemokine has no effect on postnatal tissue. There is no known migration inducing factor that induces a chemoattractant effect on the migration behavior of neuroblasts or neurons in adults after brain trauma or neurodegenerative disease.

SUMMARY OF THE INVENTION Accordingly, an object of embodiments of the present invention is to provide a novel approach to the treatment of brain injury and brain disease. Such embodiments include peptides capable of inducing one or more of nerve migration, nerve growth, nerve proliferation, nerve differentiation and / or nerve survival. These peptides are referred to herein as “nerve regeneration peptides” or “NRP”. Other embodiments include administering one or more NRPs following brain injury or during chronic neurodegenerative disease. The term “NRP” or “NRP compound” includes NRP, NRP homolog, PRP paralog, NRP ortholog and / or NRP analog. NRP alone or together with one or more other NRPs or other types of drugs that promote nerve growth, nerve migration, nerve survival, nerve differentiation and / or nerve proliferation Can be administered together.
NRPs and related peptides generally have a certain amino acid sequence that confers desired biological properties to the molecule.
Some embodiments of NRP peptides with certain domains (highlighted underline) are shown in Table 1 below.

Table 1: Nerve regeneration peptides ^*
NRP-1: YD PE AAS- APGS GNP ------ CH
NRP-2KG: KDPEARR- APGS LHP ------ C--LAA-SCSAAG
NRP-3SF: SDSFKSQ- ARG QVPPFLGGVGCPWF
NRP-4GG: GT PGR AE-AGGQVSP ------ C--LAA-SCSQAYG
NRP-5RP2: RE-- GRR D APGR A--GGGG ------ AARSVSPSP
NRP-7SW: SEPE ARR - APGR K ---- GGVVCASLAADW
NRP-8SG: SEVD ARR -AKKSLH ------- C-ILS-DTSHPRG
NRP-9SD: SEPE ARR AQGGQIPSERVLSD

In some embodiments, the NRP generally comprises a chain length of about 8 to about 25 amino acids and has a molecular weight of about 0.8 to about 2.7 kDa. In yet another embodiment, the NRP has an isoelectric point of about 6.5 to about 10.0 and further has a result selected from neuronal survival, neurite outgrowth, neuronal proliferation, neuronal differentiation and neuronal migration. It may have at least one biological property that promotes it. Furthermore, the NRP may have one or more domains indicated by the underline in Table 1 above. In some embodiments, the NRP has an [A] PG [R, S] domain together with a PE domain (eg, NRP-1 and NRP-2) and alternatively does not contain a PE domain (eg, NRP- 5, NRP-7). The presence of [A] PG [R, S] domain is desirable due to the biological properties of NRP. Furthermore, the C-terminal GG domain can confer desired neuroprotective properties to the NRP. Thus, in yet another embodiment, the NRP may have a first domain selected from the group consisting of [A] PG [R, S] domain, [A, G] RR domain and ARG domain; Has the desired biological activity. In other embodiments, desirably the NRP may have a second domain different from the first domain in addition to the first domain described above. The second domain can be a PE domain, an [A, G] RP domain, or a C-terminal GG domain. In yet another embodiment, the NRP can have a third domain of the domains described above.
Thus, in certain embodiments, NRPs contain only [A] PG [R, S] domains, other NRPs contain only ARG domains, and other NRPs contain only [G, A] RR domains be able to. Furthermore, other NRPs can have [A] PG [R, S] domains and PE domains, and other NRPs can have [A] PG [R, S] domains and [G, A] domains. Can have. Yet another NRP may have an [A] PG [R, S] domain, an [A, G] RR domain, and a PE domain.

The gene of the NRP family member contains at least one of CAAT box and TATA box, or both CAAT box and TATA box together in the promoter region. Oligonucleotides derived from NRP family members can be used to enhance NRP peptide expression in regions where such production is desired.
In another aspect, embodiments of the present invention provide a method of treatment for damaged areas of the brain as a result of head injury or chronic neurodegenerative disease. The method comprises administering one or more NRPs, NRP analogs (including peptides with structural similarity) and / or NRP prodrugs (including pro-NRP peptides) to produce neurons or neuroblasts By promoting cell migration, proliferation, survival and / or neurite outgrowth. This method of treatment is particularly useful for, but not limited to, patients with mild to severe traumatic brain injury (TBI), including neocortical injury as well as subcortical injury.
In one embodiment, NRP-2 (SEQ ID NO: 5) is encoded by a nucleic acid sequence located at base pair 77232-76768 of the reverse strand of human chromosome 13 within genomic clone bA87G1 (Sanger Sequencing Center). This peptide has functions similar to those of rat NRP-1 and can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration and neurite outgrowth.

In another embodiment, NRP-3 (SEQ ID NO: 3) is encoded by a nucleic acid sequence located at base pairs 3476-30003 according to the Double Twist annotation on the reverse strand of chromosome 3 of the human genome. . This NRP can also promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth.
In yet another embodiment, NRP-4 (SEQ ID NO: 9) is encoded by a nucleic acid located at base pair 21970003-21972239 of the forward strand of human chromosome 15, according to the NCBI human genome annotation project. Peptides translated from the nucleic acid sequence also belong to the human NRP family. Peptides encoded by this sequence can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth.
In yet another embodiment, NRP-5 (SEQ ID NO: 11) is encoded by a nucleic acid sequence located at base pair 15047153-14824042 of the reverse strand of human chromosome 7, as indicated 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 NRP is annotated with respect to a DNA sequence derived from the human genome located in the 116668725-116667697 region of the reverse strand of chromosome 6 (region from 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 another embodiment of NRP is found in rodents. Murine NRP is encoded by a nucleic acid sequence located within NCBI arachne contig_191157, which consists of 339 nucleic acids using reading frame 1. Within the overlapping region, there is a second ORF of 198 nucleic acids starting at position 29 of the annotated NRP using frame 3. This ORF encodes 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.
Yet another embodiment is NRP-8 (SEQ ID NO: 20), which is also a mouse peptide encoded by the reverse strand nucleic acid sequence in genomic clone bM344E9 of the mouse Sanger database. The protein coding sequence is annotated and is located at base pairs 5609-4052. NRP-8 can promote neuronal survival, neuronal differentiation, neuronal proliferation, neuronal migration or neurite outgrowth.
Yet another embodiment is NRP-9 (SEQ ID NO: 28). The above is a rat ortholog NRP of mouse NRP-7 (SEQ ID NO: 17), which is encoded by a nucleic acid located in the reverse strand within the following exon of the rat chromosome: exon 1, 7018685- located at positions 7022614-7022326 Exon 2 (NCBI database) located at 7018651. 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 assays that determine proliferation-inducing and migration-inducing activity. Until recently, there were few in vitro neuronal migration assays available that could detect untagged neuron migration over time. One of these bioassays monitors peri-olfactory plate cells constructed as organotypic tissue culture (“OTC”) over a 5-day time course (Fueshko and Wray, 1994). In certain embodiments of the present invention, NRP can be detected in vitro by using an adult thalamocortical OTC, neuronal migration, neuronal proliferation, neuronal differentiation, neuronal survival and / or neurites. They can be evaluated for their ability to induce elongation. These embodiments are particularly useful for the following reasons: 1) Both cultures under controlled conditions by separating the two organs physically sufficiently far apart (about 3 to about 5 mm) on the tissue culture substrate The formation of cell bridges between organs (eg thalamus and cortex) can be avoided, and 2) after birth, neuronal migration within the thalamus is substantially complete due to the time course of thalamic ontogeny Because. These bioassays would therefore be very suitable for screening and identification of neuronal migration inducers.
In certain embodiments, in vitro thalamocortical OTC assays include the advantage of revealing both radial migration within the cortex and tangential migration due to induction within the thalamus. Under in vitro control conditions, only intrinsic intracortical radial migration can be observed due to the time course of normal ontogeny of the neocortex.

In another embodiment, an in vitro bioassay comprising a cerebellar microexplant attached to a substrate is provided. These embodiments can be used to provide data regarding neuronal migration patterns, including quantification of the number of migratory neurons and migration distance for microexplants.
After only 2-3 days of culture, it is possible to observe the development of migratory chains consisting of small neurons as well as enhancement of overall cell migration. The results obtained by this assay are similar to cell chain induction within the thalamic cortical OTC.
An embodiment related to another aspect of the invention includes using NRP to treat or prevent neurodegenerative diseases and brain damage. Specifically, NRP is particularly suitable for use in brain regions that lack resting neuronal stem cells near the damaged or diseased region.
The use of NRP as a prophylactic agent can be useful in eclectic surgery such as coronary artery bypass grafting (CABG) or other surgical methods including impaired oxygen supply to the brain. Furthermore, NRP may be useful in the treatment of acute brain injury caused by, for example, stroke, trauma or other damage that impairs oxygenation to the brain or spinal cord. Furthermore, precautionary measures can be taken before radiation therapy or chemotherapy.

NRP compounds can initiate neuronal proliferation, neuronal migration, neuronal survival and / or neurite outgrowth in differentiated neural tissue after birth. These peptides are found in areas of injury in patients with mild to severe traumatic brain injury (“TBI”), including diffuse axonal injury, hypoxic ischemic brain injury and other forms of cranial brain injury. It can be used in a therapeutic method for the purpose of improving or repairing the neuron circuit. NRP compounds include treatment of nervous system infections (eg common bacterial meningitis) and those caused by stroke (ischemic infarction, embolism and hemorrhage (eg hypotension hemorrhage) and other causes ) Can be used for treatment. Furthermore, NRP compounds are also associated with neurodegenerative diseases (Alzheimer's disease, Lewy body dementia, Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis, motor neuron disease, muscular dystrophy, peripheral neuropathy, nervous system metabolism. It may be useful in the treatment of abnormalities (including glycogenosis) and other conditions in which neurons are damaged or destroyed.
In certain embodiments of the present invention, we have found that members of the NRP gene family are expressed in mammalian stem cells (both immortalized stem cells and primary stem cell cultures).

In other embodiments, we have found that NRP can promote the differentiation of stem cells into neural progenitor cells (neuroblasts). In yet another embodiment, NRPs can stimulate stem cell migration in response to chemoattractants and promote neuroblast differentiation into cells with mature neuronal morphology (eg, axons). And can further promote the growth of neurites (eg, axons and dendrites) from differentiated neuroblasts.
An embodiment of another aspect of the invention involves promoting the growth of olfactory cells using NRP.
Thus, NRP repairs damaged neurons, reforms neural tissue populations, promotes neuronal differentiation, and supports the processes necessary to promote synaptogenesis (eg, neurite outgrowth and / or neuronal differentiation) Can be an important therapeutic tool.
In other embodiments, a surgical transplant of stem cells in combination with NRP can be used to reform a population of neural tissue. The combination of stem cells or alternatively neuroblasts and NRP can promote regrowth of neural tissue. Such methods result in the remodeling of mature neural tissue and can therefore be used to treat neurodegenerative conditions. Such symptoms include hypoxia / ischemia, stroke, cardiac bypass grafting, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, and neuronal death or Other diseases with degeneration are included.
The present invention will be illustrated by describing specific embodiments thereof. Other objects, features and advantages of embodiments of the present invention will become apparent from the detailed description and drawings.

Definitions The term “homolog” includes one or more genes whose sequence is an evolutionary relationship (relationship between species (orthologs) or within species (paralogs)). Therefore, genes that have a significant association are included. Homologs also include genes that are related by being passed down from a common ancestral DNA sequence. Homologs also include relationships between genes separated by species formation events, or relationships between genes due to genetic replication events (see paralog). As used herein, the term “homolog” also includes gene products that are related to each other by evolutionary relationship. NRPs with conserved amino acid sequence domains are examples of homologs.
The term “paralog” includes a set of homologous genes that are branched from each other as a result of genetic duplication. For example, mouse alpha globin and beta globin are paralogs. As used herein, the term “paralog” also includes gene products that are related to each other by evolutionary relationship. Human NRP with a conserved amino acid sequence domain is an example of a paralog.
The term “ortholog” includes a set of homologous genes that are branched from each other as a result of species formation. For example, mouse and chicken alpha globin genes are orthologs. As used herein, the term “ortholog” also includes gene products that are related to each other by evolutionary relationships. Human and mouse NRPs with conserved amino acid sequence domains are examples of homologues.
The term “paralog peptide” includes peptides encoded by paralog nucleotide sequences.

The terms “peptide” and “protein” include polymers composed of amino acids.
The term “prodrug” includes molecules comprising propeptides that result in active NRP, active NRP analogs or active NRP paralogs as a result of enzymatic, metabolic or other processing.
The term “NRP compound” includes NRPs, NRP homologs, NRP paralogs, NRP orthologs, NRP analogs and prodrugs of NRP.
The term “NRP” is a function involving one or more of neuronal migration, neuroblast migration, neuronal proliferation, neuronal differentiation, neuronal survival and neurite outgrowth, regardless of evolutionary relationship Including peptides having
Amino acids are represented by standard symbols. In this case, alanine is “A” or “Ala”, arginine is “R” or “Arg”, asparagine is “N” or “Asn”, aspartic acid is “D” or “Asp”, cysteine Is “C” or “Cys”, glutamic acid is “E” or “Glu”, glutamine is “Q” or “Gln”, glycine is “G” or “Gly”, histidine is “H” or “ “His”, isoleucine “I” or “Ile”, leucine “L” or “Leu”, lysine “K” or “Lys”, methionine “M” or “Met”, phenylalanine “F” or “Phe”, proline “P” or “Pro”, serine “S” or “Ser”, threonine “T” or “Thr”, tryptophan “W” or “Trp” ", Tyrosine is represented by" Y "or" Tyr ", and valine is represented by" V "or" Val ". Peptides carboxy terminally amidated is indicated by -NH _2.

Nucleic acids include nucleotides including adenine (represented by “a”), thymine (represented by “t”), cytosine (represented by “c”), and guanine (represented by “g”) Consists of. Nucleotides that can be either guanine or adenine are represented by “r”, nucleotides that can be either thymine or cytosine are represented by “y”, and nucleotides that can be either guanine, adenine, cytosine, or thymine are “n”. It is represented by The polynucleotide may be DNA or RNA, and may be single-stranded or double-stranded. If the polynucleotide is an RNA polynucleotide, uracil “u” can be substituted for thymine.
“Disease” includes any non-healthy condition of the animal's CNS or peripheral nervous system, especially Parkinson's disease, Lewy body dementia, Huntington's disease, Alzheimer's disease, multiple sclerosis, motor neuron disease, muscular dystrophy , Peripheral neuropathy, nervous system metabolic abnormalities (including glycogenosis).
“Injury” includes any acute injury of an animal, particularly involving stroke, traumatic brain injury, hypoxia, ischemia, fetal distress (eg following placental detachment, umbilical cord obstruction), or Perinatal asphyxia associated with delayed intrauterine growth, perinatal asphyxia associated with failure of appropriate emergency resuscitation or aspiration, and severe CNS disorders associated with: drowning, sudden infant death, inhalation of carbon monoxide, Ammonia or other gas poisoning, cardiac arrest, coma, meningitis, hypoglycemia and status epilepticus), cerebral hypoxia episode associated with coronary artery bypass surgery, hypotension episode and hypertension crisis, brain trauma and spinal cord injury) Is included.

Description of Specific Embodiments Embodiments of the present invention include compositions and methods for the treatment of brain injury, including nerve regeneration peptides (NRP). NRPs induce neuronal migration, neurite outgrowth, nerve differentiation, nerve survival and / or nerve proliferation. NRPs can 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 due to predicted protein sequences based on previously sequenced oligonucleotides corresponding to the genes described herein. Other peptides are synthesized and at least some are provided in C-terminal amidated form. However, it will be understood that non-amidated proteins and peptides are also encompassed within the scope of the present invention.
The nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of rat NRP-1 are as follows:

9 18 27 36
5 'tat gat cca gag gcc gcc tct gcc cca gga tcg 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-NH ₂ SEQ ID NO: 2

However, because of the degeneracy of the genetic code, many codons can encode the same amino acid. Thus, various nucleic acid sequences can encode the same amino acid sequence. Each of these variations can be translated to yield SEQ ID NO: 2, and thus any of these variations are encompassed within the scope of the present invention. For example, a number of nucleic acid sequences (including the nucleic acid sequence listed in SEQ ID NO: 1) encode the rat NRP-1 amino acid sequence. The invention further includes variants of the nucleotide sequence of SEQ ID NO: 1. These include nucleic acid sequences encoding rat NRP-1 analogs and NRP-1 orthologs and / or paralogs, as well as variants that preserve the amino acid sequences encoded by the nucleic acids. By way of example only, variants of SEQ ID NO: 1 are shown below according to the genetic code for DNA (where each codon is separated from the adjacent codon by a space and the nucleic acid after the “/” is before the “/” Of nucleic acid variants):
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 / a / t 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 sequences (including display variants) can be represented using the letters r, y and n according to the definition above to provide the following sequences:
5 'tay gay ccn gar gcn gcn tcn gcn ccn ggn tcn ggn aay ccn tgy cay 3' SEQ ID NO: 3

It will be appreciated that other nucleotide sequences encoding other NRPs may vary according to the degeneracy of the genetic code. Furthermore, RNA can encode the peptide of the present invention as well as DNA, and thymine can be substituted with uracil when the nucleic acid is an RNA nucleic acid.
Human genes are annotated using human cachexic cDNA as a template (US Pat. No. 5,834,192). The survival-promoting peptide has over 96% identity with the survival-promoting peptide (Cunningham et al., 1998), and rat NRP-1 has 100% identity with the cachexia protein and has a known corresponding Only one NRP-1 homologue with cDNA. Human cachexia protein is located in the region of base pair 621841-625428 of chromosome 12 and consists of 5 exons. We compared the splicing site of the cachexic mRNA to the identified NRP human paralogue on chromosome 13 (genomic clone obtained from Sanger Sequencing Center: base pairs 77232-76768), and the coding region of the NRP-1 human orthologue (This ortholog is referred to herein as NRP-2). The nucleotide and amino acid sequences related to NRP-2 are as follows:

下線を付したヌクレオチド配列はシグナルペプチドを示す。 SEQ ID NOs: 4 and 5

Underlined nucleotide sequences indicate signal peptides.

The DNA sequence encoding the protein consists of four exons as predicted by splicing site analysis using the paralogue sequence of the human cachexia gene on chromosome 12 (cDNA of US Pat. No. 5,834,192) as a template. The chromosomal map of genomic clone bA87G1 is considered the basis for accurate exon positioning. Exon 1 is located between bp 77232-77170. Exon 2 is located between bp77088-77046. Exon 3 is located between bp77036-76824. 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 the same length as human cachexia protein, and has an overall identity of 24.5% with human cachexia protein. Sequence comparison of the signal peptide (amino acids 1-19) for extracellular localization of both proteins revealed 31.6% identity. Significantly, comparison of the first 30 amino acids of the mature (cleaved) peptide revealed 46.7% amino acid identity. Furthermore, this peptide has neuronal migration, proliferation, survival and neurite outgrowth activity similar to NRP-1 (see FIGS. 16, 17 and 18).
The second ortholog of NRP-1 is annotated. The above is encoded by a DNA sequence derived from the human genome located between base pairs 3476-33033 of the complementary reverse strand of chromosome 3 (region by the double twist human genome annotation project). This protein coding sequence consists of 5 exons arranged as follows: Exon 1, 34364-34743; Exon 2, 34729-34700; Exon 3, 33745-33596; Exon 4, 33498-33459; Exon 5, 33043-33003. This nucleotide sequence (SEQ ID NO: 6) has 333 nucleotides, and the amino acid sequence (SEQ ID NO: 7; referred to herein as NRP-3) has 111 amino acids and is represented as follows: Is:

SEQ ID NO: 6 and 7

These sequences belong to the human gene family of NRP and are referred to herein as NRP-3. This sequence has 50% identity and 67.2% similarity to the human cachexia associated protein. Furthermore, the peptide encoded by this nucleotide sequence has properties similar to NRP-1.
A third NRP-1 ortholog is annotated and is contained in the human genome-derived DNA sequence located between regions 21970003-21972239 of human chromosome 15 (which is a region from the NCBI Human Genome Annotation Project) The protein coding sequence consists of six exons arranged as follows: exon 1, 21970003-21970031; exon 2, 2197515-21970545; exon 3, 21970571-21970644; exon 4, 2100818-21970861; exon 5, 21971526-21971731; Exon 6, 21972189-21972239. This gene has been sequenced again and is now represented by SEQ ID NO: 48 and SEQ ID NO: 49 below. However, gene resequencing did not change the NRP encoded thereby. The sequence consists of 435 nucleic acids encoding 145 amino acids. This nucleotide sequence (SEQ ID NO: 8) and predicted amino acid sequence (SEQ ID NO: 9, referred to herein as NRP-4) are as follows:

SEQ ID NOs: 8 and 9

This sequence belongs to the human gene family NRP. This sequence has 45% amino acid similarity with the NRP encoded by the nucleic acid sequence located on human chromosome 13. Triplets 244-246 (amino acid positions 82); triplets 391-393 (amino acids position 131) and triplets 421-423 (amino acids position 141) encode potential N-glycosylation sites. Amino acid position 118 is x due to ambiguity in the nucleic acid sequence. The peptide, NRP-4, exhibits nerve growth promoting activity, neurite outgrowth and neuronal survival promoting activity. Note that at oligonucleotide positions 353-354, this nucleotide has not been determined and the corresponding amino acid is not known. However, following the publication of the above sequences, the exact sequences are provided and are described herein as SEQ ID NO: 48 and SEQ ID NO: 49 in Example 26. Note that this change in sequence information does not appear to affect the site associated with the neuroprotective action of the peptide derived from SEQ ID NO: 8 or SEQ ID NO: 9.
Another human ortholog of rat NRP-1 (“NRP-5”) is encoded by a DNA sequence present in the working draft of homo sapiens chromosome 7 of the NCBI database for the reverse strand. The protein coding sequence is annotated and consists of three exons with a total length of 798 nucleic acids encoding 266 amino acids. The exact arrangement of protein-encoded exons is as follows: Exon 1, 15047153-15046815; Exon 2, 14897885-14897772; Exon 3, 14824386-14824042. There is evidence in human EST (GenBank AW138864) that this mRNA is expressed. The nucleotide sequence (SEQ ID NO: 10) and amino acid sequence (SEQ ID NO: 11, NRP-5) are as follows:

SEQ ID NOs: 10 and 11

The complete protein NRP-5 consists of 266 amino acids. The annotated translated NRP amino acid sequence NRP-5 has 76% similarity to the human calcium-dependent activator protein (GenBankXP — 036915) for secretion located on chromosome 3. Furthermore, the translated NRP-5 exon 1 (339 nucleic acid) of human chromosome 7 has 95.5% homology with the translated mouse 5′EST (RIKENBB632392). This protein shares domains present in NRP-1 and other NRPs that exhibit the biological properties of neurite outgrowth, neuronal survival, neuronal proliferation and neuronal migration.
We annotated the human genome-derived DNA sequence located between regions 116668725-116667697 of the complementary reverse strand of chromosome 6 (region from the NCBI Human Genome Annotation Project). The protein coding sequence consists of three exons arranged as follows: exon 1, 116668725-116668697; exon 2, 116668333-116668305; and exon 3, 116667872-116667697. The sequence (referred to herein as NRP-6) consists of 234 nucleic acids encoding 78 amino acids. This sequence belongs to the human gene family of NRP. The highest homology found for human EST presents the uniqueness of nucleic acids 59-234 compared to the human cDNA clone GenBankCS0DK001Y119 isolated from human placental tissue. This clone is sequenced from the 3'-prime end and consists of 924 nucleic acids. Since our homolog forms a termination with a stop codon TGA after 234 nucleic acids, we are not targeting this gene product. This nucleotide sequence encoding NRP (SEQ ID NO: 12) and the amino acid sequence of this peptide (SEQ ID NO: 13, NRP-6) are as follows:

SEQ ID NOs: 12 and 13

The amino acid sequence of NRP-6 has an annotated NRP paralog on human chromosome 13, 14.1% identity and 44.9% similarity to NRP-2. This protein shares domains present in NRP-1 and other NRPs (eg, NRP-2 to 5) that have the biological properties of neurite outgrowth, neuronal survival, neuronal proliferation and neuronal migration.
Furthermore, another NRP-1 ortholog, a mouse NRP family member, was identified. This mouse NRP family member (designated here as protein 2, SEQ ID NO: 17; referred to herein as NRP-7) is located within NCBI Arachne contig_191157 and uses reading frame 1. Consists of nucleic acids. Within the overlapping region is a second ORF of 198 nucleic acids starting at position 29 of the annotated paralog using frame 3. This ORF encodes a protein (designated here as protein 1) that is highly identical to the truncated human DNA repair protein. By using the biologically active NRP peptide sequence: KDPEARRAPGSLHPCLAASCSAAG-NH2 (SEQ ID NO: 18) and utilizing the search paradigm tBLASTI, we obtained blast hits in the mouse EST RIKEN database. This 5′-producing mouse EST has accession number GenBankAK012518 and the following sequence (SEQ ID NO: 14):

5'- GGCAGCCTCGAGATGGGGAAGATGGCGGCTGCTGTGGCTTCATTAGCCACGCTGGCTGCAGAGCCCAGAGAGGATGCTTTCCGGAAGCTTTTCCGCTTCTACCGGCAGAGCCGGCCGGGGACAGCGGACCTGGGAGCCGTCATCGACTTCTCAGAGGCGCACTTGGCTCGGAGCCCGAAGCCCGGCGTGCCCCAGGTAGGAAAGGAGGAGTAGTGTGTGCCAGCCTAGCGGCCGACTGGGCCACCCGAGACTGGGCCGCCTCCGGGCCGGCTTTGGAGGGAAGCCCCTGCTGGGCCTGTCCAGTGAGCTGTAATGTCGAGCGATGAGCGACCAGCTGCCTCGCTGTCCCAACGCTCTGGCCACGGCTTGTGCCTTGCCGCCATTTCCCCCAACCCACGCGGGCCACGGCTTGTGCCCTGCCGCCATTTCCCCCAACCCACGCGACCTTGCTC - 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) revealed the following protein sequence (SEQ ID NO: 15):
MGKMAAAVASLATLAAEPREDAFRKLFRFYRQSRPGTADLGAVIDFSEAHLARSPKPGVPQVGKEE SEQ ID NO: 15
This sequence has 82% amino acid sequence homology (identity and chemical similarity) with the human alkylated DNA repair protein of GenBank accession number Q13686. The mouse type is truncated at the C-terminus and only has 66 amino acids out of 389 amino acids of the human DNA repair protein.
Protein 2 reading frame 1
Long ORFs with as many as 323 nucleic acids can be found in frame 1 of the EST sequence. Subsequently, we annotated the 5 ′ end of the ORF of the 323 nucleic acid in the mouse genome, and found a novel gene located between 23970 and 24374 of the mouse Arachne contig_191157 sequence in the NCBI database. This protein coding sequence consists of two exons with a total length of 339 nucleic acids encoding 113 amino acids. The position of exon 1 is 23970-23990 and the position of exon 2 is 24057-24374. The nucleotide sequence (SEQ ID NO: 16) and amino acid sequence (SEQ ID NO: 17, NRP-7) of this mouse NRP ortholog of rat NRP-1 are as follows:

SEQ ID NO: 16 and 17

  The expressed amino acid sequence of complete NRP-7 contains 113 amino acids (SEQ ID NO: 17). Another version of NRP-7 is also another splice form, which includes another 66 nucleotides after position 21 of SEQ ID NO: 16 (SEQ ID NO: 35), which is a long NRP having 135 amino acids -7 (“NRP-7 long”) (SEQ ID NO: 36).

The protein function programming tool SMART predicts a single peptide sequence of 28 amino acids. This protein has 13.6% identity and 23.6% similarity to the NRP ortholog of human chromosome 13, and also has neuronal survival, migration, proliferation and growth activity similar to NRP-1.
The second mouse NRP family member is located on the complementary reverse strand in the genomic clone bM344E9 of the mouse Sanger database. By using the search program tBLASTN using the biologically active NRP peptide sequence: KDPEARRAPGSLHPCLAASCSAAG-NH ₂ (SEQ ID NO: 18), we have similarity within the genomic clone bM344E9 in the mouse genomic Sanger database Got the area. The protein coding sequence is annotated and consists of 5 exons. The above is a nucleic acid having a total length of 423 encoding 141 amino acids. The arrangement of the code exons is as follows: 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 amino acid sequence (SEQ ID NO: 20) of the mouse ortholog of rat NRP-1 (referred to herein as NRP-8) are as follows:

SEQ ID NO: 19 and 20

The expressed amino acid sequence of NRP-8 contains 141 amino acid residues. The asparagine residues at positions 112-114 are presumed to be N-glycosylated due to the presence of the N-glycosylation consensus sequence. This new mouse NRP-1 ortholog, NRP-10, is 35.5% homologous to the human NRP ortholog located on chromosome 13 (NRP-2), and further to the mouse NRP-1 ortholog located in the Arachne contig of NCBI. It has 28.9% homology. Furthermore, the peptide comprises an amino acid sequence domain similar to that present in NRP-1 or other NRP peptides, and the peptide further comprises promoting neuronal migration, proliferation, survival and / or neurite outgrowth. Has biological properties.
The nucleotide sequence (SEQ ID NO: 27) and amino acid sequence (SEQ ID NO: 28) of NRP-9 (rat ortholog of mouse NRP-7) are as follows:

SEQ ID NOs: 27 and 28

  In addition to the NRP compounds described above, we have identified other genes with NRP-like peptide domains that may also be useful for NRP expression. These include genes derived from mycobacteria and tumor cells. A recently published paper disclosed a PE multigene family of Mycobacterium tuberculosis containing a consensus sequence (PE_PGRS), which is similar to the sequence we proposed (PGR / S). They also state that these proteins are released by the bacteria in the host and promote bacterial survival. There are examples where the PE_PGRS consensus sequence that they provided in the paper is found. The amino acid sequence (SEQ ID NO: 21) of the Rv1818c gene product of Mycobacterium tuberculosis is shown below:

msfvvtipea 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 gligdggdng aggngtgakg

  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 kggwfgkhrg qggsnpkfen
iaeglralla rshverttde gtwvagvfvy ggsktslynl rrgtalaipq crltplsrlp
fgmapgpgpq pgplresivc yfmvflqthi faevlkdaik dlvmtkpapt cnirvtvcsf
ddgvdlppwf ppmvegaaae gddgddgdeg gdgdegeegq e SEQ ID NO: 22
(From MJ Brennan and G. Delogu, The PE multigene family: a “molecular mantra” for mycobacteria. Trends in Microbiology 5: 246-249 (2002))

It will be appreciated that the complete sequence from NRP-1 to NRP-9 need not be used. Rather, a peptide fragment of about 8 amino acids can be used in accordance with embodiments of the present invention. Given the consensus sequence domains identified herein, synthetic peptides can be created or the naturally occurring NRP can be truncated to obtain biologically active peptide moieties. Methods for shortening the expressed peptide (eg, using synthetic DNA) or enzymatic modification are known in the art.
One embodiment of the invention is a 24mer fragment of NRP-2 (SEQ ID NO: 5) comprising the sequence KDPEARRAPGSLHPCLAASCSAAG-NH ₂ (NRP-2 segment KG; SEQ ID NO: 18).
Yet another embodiment of the invention is a 19mer fragment of NRP-2 (SEQ ID NO: 5) comprising the sequence KDPEARRAPGSLHPCLAAS-NH ₂ (NRP-2 segment KS; SEQ ID NO: 23).
Yet another embodiment of the present invention is a 24-mer form of NRP-7 (SEQ ID NO: 17 or SEQ ID NO: 36) comprising the sequence SEPEARRAPGRKGGVVCASLAADW-NH ₂ (NRP-7 segment SW; SEQ ID NO: 24).
Yet another embodiment of the invention comprises the sequence SDSFKSQARGO-NH ₂ (NRP-3 segment SQ; SEQ ID NO: 25) (located between amino acids 11-23 of NRP-3 (SEQ ID NO: 7)). 11mer peptide.

Another embodiment of the invention comprises the sequence GTPGRAEAGGQ-NH ₂ (NRP-4 segment GQ; SEQ ID NO: 26) (amino acids 22-32 of annotated NRP-4 (SEQ ID NO: 9)). 11mer peptide located between).
Yet another embodiment of the present invention is a 25mer fragment of NRP-4 (SEQ ID NO: 9) comprising the sequence GTPGRAEAGGQVSPCLAASCSQAYG-NH ₂ (NRP-4 segment GQ; SEQ ID NO: 29).
Yet another embodiment of the present invention is a 13mer fragment of NRP-5 (SEQ ID NO: 11) comprising the sequence REGRRDAPGRAGG-NH ₂ (NRP-5 segment RG; SEQ ID NO: 30).
Yet another embodiment of the invention is a 24-mer fragment of NRP-8 (SEQ ID NO: 20) comprising the sequence SEVDARRAKKSLHCILSDTSHPRG-NH ₂ (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 the sequence SDSFKSQARGQVPPFLGGVGCPWF-NH ₂ (NRP-3 segment SF; SEQ ID NO: 32).
Yet another embodiment of the invention is an 8mer fragment of NRP-5 (SEQ ID NO: 11) comprising the sequence REGRRDAP-NH ₂ (NRP-5 RP; SEQ ID NO: 33).
Yet another embodiment of the present invention is a 21mer fragment comprising the sequence SEPEARRAQGGQIPSERVLSD-NH ₂ (NRP-9 segment SD; SEQ ID NO: 34), which sequence is amino acid residue 88 of NRP-9 (SEQ ID NO: 28). Located between -108.
Yet another embodiment of the present invention is a 9mer peptide comprising the sequence PGRAEAGGQ-NH ₂ (NRP-4 segment PQ; SEQ ID NO: 43), wherein the sequence is annotated NRP-4 (SEQ ID NO: 9) Is located between amino acids 24-32. Still other embodiments are described elsewhere herein.

Use of NRP compounds The invention therefore, NRP, includes peptides encoded by the NRP, homologs of NRP, orthologs or paralogs, the embodiments prodrugs analog and NRP of NRP, wherein prodrugs of NRP is an enzyme In particular, a molecule that can be metabolically or otherwise modified to become an NRP, NRP homolog, NRP paralog, NRP ortholog or NRP analog. Such molecules are collectively referred to as “NRP compounds” or “NRP”. NRP compounds may be encoded by nucleotide sequences. The nucleotide sequence may be DNA or RNA, and may be single-stranded or double-stranded. It will be understood that the present invention includes sequences complementary to said sequences as well as the sequences themselves disclosed in this application. Furthermore, it will be understood that other splice forms of NRP may exist. In such cases, other splice forms of NRP RNA, as well as the proteins and peptides that they can encode, are also considered to be part of the present invention.
As indicated above, embodiments of the present invention show that NRPs can induce neuronal and neuroblast proliferation, migration, differentiation, neurite generation and further protect neurons from damage caused by nerve damage It is based on the inventors' surprising discovery that they can. Nerve cell proliferation and migration to the injured area caused by acute brain injury or chronic neurodegenerative disease can result in improved neurological function. Furthermore, NRP compounds can be used to treat a variety of abnormalities and symptoms where brain tissue is degenerated, at risk of degeneration or death, or is already dead.
Cells can also stimulate NRP production using NRP oligonucleotides after transfection. In some cases, transfection can be performed as a replicable vehicle, and in other cases, NRP oligonucleotides can be introduced as naked DNA.

Abnormalities and symptoms that can be treated with NRP
Abnormalities and symptoms for which NRP compounds may be useful include:
Central nervous system infections (including bacterial, fungal, spirochete, parasitic infections) and sarcoids (including purulent infections, acute bacterial meningitis, leptitis);
Cerebrovascular disease (stroke, ischemic stroke, atherosclerotic thrombus, lacune, embolism, hypertensive bleeding, aneurysm rupture, vascular malformation, transient ischemic stroke, intracranial hemorrhage, idiopathic subarachnoid Including bleeding, hypertensive encephalopathy, inflammatory diseases of the cerebral arteries such as reduced perfusion caused by heart failure (possibly resulting from coronary artery bypass surgery), and other forms of cerebrovascular disorders);
Brain skull trauma (including skull base fracture and intracranial nerve injury), carotid sinus fistula, pneumoencephalopathy, aneurysm and rhinorrhea, brain contusion, traumatic intracerebral hemorrhage, acute brain swelling in children;
Demyelinating diseases (including optic neuromyelitis, acute disseminated encephalomyelitis, acute and subacute necrotizing hemorrhagic encephalitis, Schirder diffuse encephalopathy, and multiple sclerosis associated with peripheral neuropathy);
Degenerative disorders of the nervous system (symptoms of one or more progressive dementias, diffuse brain atrophy, non-Alzheimer-type diffuse cortical atrophy, Lewy body dementia, Pick's disease, frontotemporal dementia, thalamic degeneration, non Including Huntington's chorea and dementia, corticospinal degeneration (Jakob disease), dementia-Parkinson's disease-amyotrophic lateral sclerosis complications (Guamanina et al.));
Acquired metabolic disorders of the nervous system (confusion, stupor or coma-ischemia-hypoxia, hypoglycemia, hyperglycemia, hypercapnia, liver failure and Reye syndrome as symptoms including one or more Metabolic disease presented, metabolic disease presented as progressive extrapyramidal syndrome, cerebellar ataxia, hyperthermia, metabolic disease presented as celiac-sprue disease, metabolic disease causing psychosis or dementia ( Including Cushing's disease and steroid brain disorders), including thyroid psychosis and hypothyroidism, pancreatic brain disorders);
Diseases of the nervous system due to nutritional deficiencies;
Alcohol and alcohol addiction;
Drugs and other chemicals (opiate and synthetic analgesics, sedatives, stimulants, psychotropic drugs, bacterial toxins, phytotoxins, snake bites and stings wound poisons, heavy metals, industrial toxins, antitumor agents and immunosuppressants, Nervous system abnormalities caused by thalidomide, aminoglycoside antibiotics (otoxins) and penicillin derivatives (convulsions), cardioprotectants (including β-blockers, digitalis derivatives and amiodarone).

As demonstrated by the above list, the compositions and methods of the present invention may be useful for the treatment of human nerve damage and disease. More generally, the compositions and methods of the present invention provide acute brain injury (diffuse axonal injury, perinatal hypoxia-ischemic injury, traumatic brain injury, stroke, ischemic infarction, embolism and hypertension). Bronchial bleeding; exposure to CNS toxicants, cranial nervous system infection (eg bacterial meningitis); including metabolic disorders (eg, including hypoxia-ischemic encephalopathy, peripheral neuropathy and glycogenosis) But with neurological damage as a result of, but not limited to, chronic nerve injury or neurodegenerative diseases (including multiple sclerosis, Lewy body dementia, Alzheimer's disease, Parkinson's disease and Huntington's disease), Useful in the treatment of human patients suffering from (but not limited to). Patients with such diseases or injuries can benefit greatly from treatment protocols that can initiate neuronal proliferation and migration as well as neurite outgrowth.
More generally, the present invention applies to the induction of neuronal and neuroblast migration to injury areas after trauma, exposure to toxins, asphyxia or hypoxia-ischemic injury.
NRP compounds (including prodrugs containing NRP-1, its orthologs, analogs, paralogs and identified NRP peptide domains) can be used to promote neuronal and neuroblast migration.

However, although NRP can be used advantageously, it is not intended to exclude administration of other forms of NRP compounds. For example, human paralogue or peptide fragments of NRP can also be administered instead of NRP. By way of example, by administering a prodrug form of NRP comprising NRP and a carrier (NRP and said carrier are linked by a bond that is sensitive to cleavage or digestion in the patient's body), The effective amount of can be increased. Any suitable linkage that is cleaved or digested to release NRP after administration can be utilized.
Another suitable therapeutic method is a transplant which is a cell line capable of expressing NRP or an analog, paralog or propeptide of NRP in an active form within the central nervous system, or a transplant comprising said cell line Through to increase the patient's NRP level.
NRP can be administered as part of a medicament or pharmaceutical preparation. This requires combining the NRP compound with any pharmaceutically suitable carrier, adjuvant or excipient. Furthermore, the NRP compound may be used with other non-NRP neuroprotective agents, proliferative agents or other agents. Of course, the choice of carrier, adjuvant or excipient will generally depend on the route of administration used.
The route of administration can vary widely. NRP can be administered in various ways, i.e. intraperitoneally, intravenously or intraventricularly. Peripheral application can also be a selection method because there is no direct interference with the central nervous system.

Any peripheral route of administration known in the art can be used. These include parenteral routes such as injection into the peripheral circulation, subcutaneous, intraorbital, intraocular, intrathecal infusion (using sustained release devices or minipumps such as osmotic pumps or skin patches), implants, Aerosol, inhalation, severing, intraperitoneal, intracapsular, intramuscular, intranasal, oral, buccal, pulmonary, rectal or vaginal routes are included. The composition is administered parenterally to a human or other mammal in a therapeutically effective amount (eg, an amount that eliminates or reduces lesions in the patient) to provide treatment for the neurological disorders described above. Can be formulated for this purpose.
One route of administration includes subcutaneous injection (eg, dissolved in 0.9% sodium chloride) and oral administration (eg, in a capsule).
It will also be appreciated that in some cases it may be desirable to administer the NRP compound directly to the patient's CNS. This can be done by any suitable direct route of administration. Specific examples include lateral ventricular injection or administration by shunt into the lateral ventricle of a patient's brain that has been surgically inserted.

Determination of NRP dosage Determination of the effective amount of NRP to be administered is within the skill of the artisan and will be routine for those skilled in the art. In certain embodiments, the amount of NRP used can be estimated in an in vitro test using the assay system described herein. The final amount administered will depend on the route of administration, the NRP used, and the nature of the neurological disease or condition to be treated. Suitable dosage ranges are, for example, from about 0.01 mg to about 1 mg per 100 g body weight, and alternatively from about 0.06 μg to about 0.6 mg of NRP per 100 g body weight, wherein the dosage is administered centrally.
For inclusion in medicine, NRP can be synthesized directly by conventional methods, such as the stepwise solid phase synthesis method of Merrtfield et al. (J. Am. Chem. Soc. 15: 2149-2154 (1963)). . Such peptide synthesis methods are well known in the art and are described, for example, in the following literature: Fields and Colowick, 1997, Solid Phase Peptide Synthesis (Methods in Enzymology, vol. 289), Academic Press, San Diego, CA. Alternatively, the synthesis can include a commercially available peptide synthesizer, such as an Applied Biosystems model 430A.
As a general proposition, the total pharmaceutically effective amount of NRP-1 (SEQ ID NO: 2) administered parenterally is within the range measurable by a dose response curve. A range is from about 0.06 mg to about 0.6 mg per 100 g body weight. For example, blood NRP-1 (SEQ ID NO: 2) can be measured in the body fluid of a mammal to be treated to determine the dose. Alternatively, the NRP-1 (SEQ ID NO: 2) compound can be administered to a patient in increasing amounts and the patient's serum level of NRP-1 (SEQ ID NO: 2) can be checked. The amount of NRP-1 (SEQ ID NO: 2) to be used can be calculated on a molar basis based on the serum levels of these NRP-1 (SEQ ID NO: 2).

In particular, one method of determining an appropriate dose of the compound involves measuring NRP levels in biological fluids (eg body fluids or blood). Such level measurements can be performed by any method (including RIA and ELISA). After measuring the NRP level, the fluid is contacted with the compound using a single dose or multiple doses. After this contacting step, the NRP level in the fluid is measured again. If the NRP level of the fluid is reduced by an amount sufficient to produce the desired efficacy for the purpose for which the molecule is administered, the dosage of the molecule can be adjusted to produce maximum efficacy. This method can be performed in vitro or in vivo. This method can be used in vitro, for example, withdrawing the fluid from the mammal, measuring NRP levels, and administering the compound herein to the mammal in single or multiple doses (ie, the contacting step is directed to the mammal). This can be done after the NRP level has been measured again with fluid drawn from the mammal.
The NRP compound can be suitably administered by a sustained release system. Suitable examples of sustained release compositions include semipermeable polymer matrices that are shaped as shaped articles (eg, films or microcapsules). Sustained release matrices include polypeptide lactide (US Pat. No. 3,773,919, EO58,481), poly (2-hydroxyethyl methacrylate) (Langer et al., 1981), ethylene vinyl acetate (Langer et al., Supra). Or poly-D-(-)-3-hydroxybutyric acid (EP133,988). Sustained release compositions also include liposome-binding compounds. Liposomes containing this compound are produced by methods known to those skilled in the art. The above is carried out by: DE 3,218,121; Hwang et al., 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; -118008, U.S. Pat. Nos. 2,485,045 and 4,544,545, and EP 102,324. In some embodiments, the liposomes are small (about 200 angstroms, or about 200 angstroms to 800 angstroms) in a single lamina type, in which case the lipid content exceeds about 30 mol percent cholesterol, the selected ratio being the most Adjusted for effective treatment. All US patents cited herein (above and below) are hereby incorporated by reference.

For example, a PEGylated peptide having a longer lifetime than a non-PEGylated peptide by a composite technique described in WO95 / 32003 (published on November 30, 1995) can also be used.
For parenteral administration, the dosage is from about 0.01 to about 1 mg / 100 g body weight, alternatively from about 0.06 μg to about 0.6 mg of NRP compound per 100 g body weight. In some embodiments, the compound is generally mixed with each compound of the desired purity and is toxic or non-toxic to the recipient at a pharmaceutically or parenterally acceptable carrier (ie, dosage and concentration used). Can be formulated in an injectable unit dosage form with those compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be deleterious to polypeptides. It will be appreciated that the above dosages are not limiting. Other doses outside the above ranges can also be determined by one skilled in the art.
In some embodiments, the compounds can be made by contacting uniformly and intimately with a liquid carrier, or a finely divided solid carrier, or both. Subsequently, if desired, the product can be molded into the desired formulation. In some embodiments, the carrier is a parenteral carrier and alternatively is a solution that is isotonic with the blood of the recipient. Examples of such media as carriers include water, saline, Ringer's solution, buffer solution, and dextrose solution. Non-aqueous media such as fixed oils and ethyl oleate are also useful herein.

The carrier 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 used and include, by way of example: buffers (eg, phosphoric acid, citric acid, succinic acid, acetic acid and other organic acids or Their salts); antioxidants (eg ascorbic acid); low molecular weight (less than 10 residues) polypeptides (eg polyarginine) or tripeptides; proteins (eg serum albumin, gelatin or immunoglobulins); hydrophilic polymers (eg Polyvinyl pyrrolidone); glycine; amino acids (eg, glutamic acid, aspartic acid, histidine, or arginine); monosaccharides, disaccharides, and other carbohydrates (cellulose or derivatives thereof, glucose, mannose, trehalose, or dextroline); chelating agents ( Sugar alcohol (eg mannitol or Rubitoru); counterions (such as sodium); non-ionic surfactants (e.g. polysorbate, poloxamer or polyethylene glycol (PEG)); and / or neutral salt (e.g. NaCl, KCl, etc. MgCl _2, CaCl _2).
The NRP compound can desirably be formulated in a medium as described above at a pH of about 4.5 to about 8. It will be appreciated that the use of certain of the aforementioned vehicles, carriers or stabilizers will result in the formation of a salt of the compound. The final preparation may be a stable liquid or a lyophilized solid.

In other embodiments, adjuvants can be used. Typical adjuvants that can be incorporated into tablets, capsules, etc. are binders (eg gum arabic, corn starch or gelatin); vehicles (eg microcrystalline cellulose); disintegrants (eg corn starch or alginic acid); A sweetener (eg, magnesium stearate); a sweetener (eg, sucrose or lactose); and a fragrance (eg, peppermint, citrus or cherry). When the dosage form is a capsule, in addition to the materials described above, liquid carriers such as fatty oils can also be included. Various types of other materials can be used as coatings or as physical form modifiers of the dosage unit. A syrup or elixir may contain the active compound, sweetening agents (eg sucrose), preservatives (eg propylparaben), coloring agents and flavoring agents (eg cherry). Sterile compositions for injection can be formulated according to normal pharmaceutical practice. For example, it may be desirable to have a solution or suspension in the active compound medium such as water or a naturally occurring vegetable oil such as sesame oil, peanut oil or cottonseed oil or a synthetic fatty medium such as ethyl oleate. it can. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.
Desirably, the NRP compound used for therapeutic administration can be sterilized. Sterilization can be easily performed by filtration through a sterile filtration membrane (eg, a membrane having a pore size of about 0.2 microns). The therapeutic composition can generally be placed in a container with a sterile access port (eg, a vial with a stopper that can be pierced by an intravenous solution bag or hypodermic needle).
In other embodiments, the NRP compound can be stored in unit dose or multi-dose containers (eg, enclosed ampoules or vials) as an aqueous solution or a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10 mL vial is filled with 5 mL of sterile filtered aqueous solution of compound (0.01% w / v) and the resulting mixture is lyophilized. Infusion solutions can be prepared by reconstitution of the lyophilized compound with antibacterial water or other suitable solvent.

Gene Therapy In another embodiment of the invention, the method of treatment comprises gene therapy for organ therapy, which uses a nucleic acid encoding an NRP compound. In general, gene therapy can be used to increase (or overexpress) NRP levels in an organ. Examples of nucleotide sequences include SEQ ID NOs: 1, 3, 4, 6, 8, 10, 12, 16, 19, 27 or 35, or portions thereof encoding peptides with NRP consensus domains and biological properties. Is included. It will be appreciated that other sequences encoding pro-NRP can also be used (pro-NRP can yield biologically active NRP upon cleavage).
Any suitable method can be used to transfect an organism with a sequence encoding NRP. For example, in vivo and ex vivo methods can be used. For in vivo delivery, nucleic acid (alone or combined with a vector, liposome, precipitate) is directly to an organism (eg, a human patient), and in some embodiments, expression of an NRP compound is desired. The site can be injected. For ex vivo treatment, the cells of the organism are removed, nucleic acids are introduced into these cells, and the modified cells are administered directly to the organism or encapsulated in, for example, a porous membrane which is then applied to the patient. (See, eg, US Pat. Nos. 4,892,538 and 5,283,187).
We note here that when cultured cells are capable of expressing NRP, and when these NRP-expressing cells are incubated with neurons that are susceptible to harmful injury, NRP is expressed and secreted into the culture medium, It has been shown that neurons can be protected from harmful injury. This surprising finding supports a therapeutic method of treating neurodegeneration by gene transfer and cell transplantation.
There are a variety of techniques available for transferring nucleic acids into living cells. These techniques may vary depending on whether the nucleic acid is transferred to cultured cells in vitro or to the host cells of interest in vivo. Techniques suitable for in vitro transfer of nucleic acids into mammalian cells include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation methods, and the like. A commonly used vector for ex vivo delivery of genes is a retrovirus.

In certain embodiments, in vivo nucleic acid transfer techniques include, for example, transfection with viral vectors (eg, adenovirus, herpes simplex virus I, or adeno-associated virus) and lipid utilization systems. Useful qualities for lipid-mediated gene transfer include N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium (DOTMA), dioleoyl fatidylethanolamine (DOPE) and 3-β [N- (N ′, N′-dimethylaminoethane) carbomoyl] cholesterol (DC-Chol). In some situations, it may be desirable to provide a source of nucleic acid with an agent that directs the nucleic acid-containing vector to a target cell. Such “target-inducing” molecules include cell surface membrane proteins or antibodies specific for target cells, ligands for receptors on target cells, and the like. When liposomes are used, proteins that bind to endocytosis-related cell surface membrane proteins can be used for target induction and / or promotion of uptake. Examples of such proteins include capsid proteins and their fragments that have affinity for specific cell types, antibodies against proteins that undergo internalization in cycling, and enhanced intracellular half-life targeting intracellular localization Protein to be included. In other embodiments, receptor-mediated endocytosis can be used. Such methods are described, for example, in the following: Wu et al., 1987; and Wanger et al., 1990. For a review of previously known gene marking and gene therapy protocols see the literature (Anderson, 1992). See also WO 93/2563 and the references cited above.
Kits are also contemplated within the scope of the present invention. A typical kit includes a container (in some embodiments, a vial) for an NRP formulation, the container comprising one or more NRP compounds in a pharmaceutically acceptable buffer and a user. Includes instructions for use (eg, product inserts or labels) directing the use of the pharmaceutical formulation.

Examples The following examples are provided to illustrate certain embodiments of the invention. It will be readily appreciated that other embodiments can be devised and still be within the scope of the present invention. Any of these other embodiments are considered to be within the scope of the present invention.

Example 1: Identification of the NRP gene To identify mammalian migration inducers that are effective for neuronal survival, proliferation and differentiation, we have identified the cDNA of human cachexia-related protein and its encoded A 16-mer cachexia fragment was used as a template to screen rat and mouse genomes to identify novel NRP homologues.
Identification of the NRP gene involves obtaining total RNA from various cell sources (in vivo tissue, neural stem cell culture). RNA was extracted using the Roche Total RNA Isolation Kit. Complementary DNA (cDNA) was synthesized using Superscript RT II, followed by multiplex PCR amplification of mouse NRP gene and beta actin using the following primers: mouFS NRP forward primer: 5 'AACGGAATGAATCGAAACCC 3' (SEQ ID NO: 37); mouFS Rev primer: 5 ′ CGCTCGACATTACAGCTCA 3 ′ (SEQ ID NO: 38); mouse β-actin Fwd: 5 ′ GAAAGGGTGTAAAACGCAGC 3 ′ (SEQ ID NO: 39); mouse β-actin Rev: 5 ′ GGTACCACCATGTACCCAGG 3 '(SEQ ID NO: 40). Fragments with the correct size were gel purified, cloned directly into the pGEM vector, and transformed into a competent strain of E. coli, DH5α. Transformed cell colonies were screened for the presence of the NRP gene fragment and the plasmid sequence from the positive colonies was determined. The structure of the gene encoding the NRP gene is shown in FIG. 1A.

Characterization of the NRP protein domain : NRP has a very significant score when analyzing the protein sequence using the SecretomeP server (Technical University of Denmark), probably like FGF-1, FGF-2 It will be secreted by a non-classical route. The high number of positively charged amino acid residues and the overall number of atoms in the N-terminal NRP led to this expectation (Bendsten et al., 2004).
A single trefoil domain sequence motif is predicted within the mouse NRP sequence, which has 52.5% homology with the human pS2 protein (Jakowlew et al., 1984) (FIG. 1D). A putative NRP trefoil domain is similar or identical in 9 of the 15 conserved amino acids within the consensus sequence of said trefoil domain (database SMART). This 60% consensus value is also the threshold for accepting NRP as a member of the trefoil factor family. Trefoil domains have been implicated in protein-protein interactions and have been suggested to act through the cyclooxygenase 2 (COX-2) and thromboxane A2 receptor (TXA2-R) activation pathways (Rodrigues et al., 2003) . Human Ps2 protein belongs to trefoil factor family 1 (TFF-1), and its relationship with chemoattraction of breast cancer cells (Prest et al., 2002) is considered by the signaling action in the REK1 / 2 pathway (Graness et al. , 2002).
The mouse NRP (NRP-7 segment SW) domain that promotes neuronal survival, proliferation, migration and differentiation is located C-terminal to the trefoil domain, and for rat NRP-9, C-terminal (FIG. 1B). (See aligned 21mer and 24mer sequences).

Homology between NRP, cachexia-related protein and SDF-1 : Despite the surprisingly similar biological activity between NRP and SDF-1α, both peptides have a moderate 32.6% homology It only showed similarity (Figure 1E).
The first 9 amino acids of mature SDF-1 show residual chemoattractant activity, which is almost 100 times lower than CDF-1α (Loetscher et al., 1998). The N-terminal 11 amino acids of the mouse 24mer peptide (NRP-7 segment SW; SEQ ID NO: 24) are aligned with greater than 50% homology with the start of mature SDF-1. Protein domains exhibiting this association indicate that cachexia proteins, NRP and SDF-1α share biologically active domains important for neuronal survival, proliferation, migration and differentiation. The biologically active region is located at the N-terminus of the mature protein for cachexia-related protein and SDF-1, but for NRP this region is located C-terminal to the trefoil domain.

Example 2: Cerebellar microexplant: Neuroprotection and neuron proliferation method Laminar cerebellar cortex was removed from a child of P3 / 4, P7 / 8 rats and ground through a metal sputum with a pore size of 125 μm and uniformly microexposed. Got the plant. After centrifugation at 61g for 3 minutes, the pellet is suspended in StartV medium (Biochrom) and the suspension is seeded on a poly-D-lysine-coated coverslip in a 6-well plate and incubated for 3 hours to adhere 1 mL of start V was then added per well. Glutamate / 3-NP and NRP were also added as previously described. The explants were cultured for 48-72 hours at 34 ° C. under 5% CO ₂ and 100% humidity. For growth rate measurements, BrdU was added at the start of the culture and cells per microscopic field were counted 48-72 hours later.

Result :
(A) Neuroprotection : Nanomolar NRP-4 segment GQ (SEQ ID NO: 26) resulted in 50% survival after severe injury (Figure 2).
(B) Neuronal proliferation : NRP-7 segment SW (SEQ ID NO: 24) increased the proliferation rate to over 200% in these cultures (Figure 3). Proliferating cells of the cerebellar microexplants were resistant to excitotoxic and oxidative stress compared to the effects of vehicle alone.

Example 3: Contact chemotaxis migration assay For the chemoattractant activity / chemoattractant activity of mouse neural stem cells, EGF-dependent immortalized mouse neural stem cells MEB5 and wild-type PC-12 cells, NRP was determined by contact chemotaxis migration assay as described below. Tested.
Method :
Initial NRP coating : Control wells of a transwell plate (Corning) with a pore size of 12 μm were coated with 1.5 mL of BSA / PBS medium. The remaining plates were coated with various concentrations of NRP (prepared with PBS containing 10 μg / mL BSA) ranging from 1-100 ng / mL. Subsequently, the plates were coated at 37 ° C. for 1 hour. Subsequently, the wells were washed twice with 1 mL of sterile PBS.
Extracellular matrix coating : Laminin (10 μg / mL) for MEB-5 cells, PDL (50 μg / mL) + Matrigel for mouse primary stem cells, and Fibronectin (25 μg / mL) + Matrigel for PC-12 cells Used as an extracellular matrix (ECM). All ECM compounds were diluted with PBS. 1.5 mL of ECM per well was incubated at room temperature for 2 hours. Subsequently, the wells were washed once with 1 mL of serum-free medium (eg, NB / B27) followed by 1 mL of PBS.
Insert coating : The insert was coated with a 5 μg / mL PDL / PLL mixture (in PBS). Subsequently, the insert was washed with MiliQ water.
Transfer to culture and seeding of cells : Appropriate cultures were transferred to 12-well plates (MBE-5 cells: DMEM high glucose + N2 (Invitrogen growth medium supplement) + 10 ng / biotin + 2 mM L-glutamine; primary Stem cells: Euroclon NSA culture solution; and PC-12 cells: NB / B27 times solution). Subsequently, the plates were incubated at 37 ° C. with 5% CO ₂ and seeded with 1-2 × 10 ⁵ cells. Plates were fixed in vitro for 1-2 days (DIV).
Fixing : The insert was discarded and the wells were fixed with serial dilutions of PFA (0.4, 1.2, 3 and 4%) for 3-5 minutes for each dilution. Wells were washed and stored for 3-5 minutes for each dilution in serial dilutions of PFA (0.4, 1.2, 3 and 4%). Wells were washed in PBS until counting. All cells that showed neurite outgrowth and migrated to the bottom chamber were counted as migrating cells.

Result :
10 ng / mL NRP-4GG (SEQ ID NO: 29) migrated more than 195% of MEB cells to the bottom of the culture dish compared to BSA-vehicle alone (FIG. 4A). In the presence of NRP-4GG (SEQ ID NO: 29) peptide, more than 93.7% of E14 cells migrated compared to BSA-vehicle alone (FIG. 4B). In the presence of NRP-2KS (SEQ ID NO: 23), more than 109% of MEB-5 cells migrated to the bottom compared to BSA-vehicle alone (FIG. 4B). In NRP-7SW (SEQ ID NO: 24) treated wells, E14 cell densities greater than 35% were obtained compared to BSA-vehicle alone (FIG. 4D). In the presence of NRP-4GG (SEQ ID NO: 29) peptide, more than 80.8% of PC-12 cells migrated to the bottom compared to BSA-vehicle alone (FIG. 4E). NRP-7SW (SEQ ID NO: 24) migrated more than 333% PC-12 cells compared to BSA-vehicle alone (FIG. 4F).

Example 4: Differentiation assay for neural stem cell culture and axonal outgrowth
NSA stem cell culture was purchased from Euroclone (Italy). Neurobasal, DMEM / F12 media, N2 and B27 supplements were all obtained from Life Technology. Anti-βIII-tubulin antibody was purchased from Sigma. Cy3-conjugated goat anti-mouse antibody was purchased from Amersham and Syto21 was purchased from Molecular Probes. NRP-2KS (SEQ ID NO: 23) and NRP-7SW (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 the forebrain of E15 C3H mice and were cultured as neurospheres in the presence of 20 ng / mL EGF and 10 ng / mL bFGF as described in the literature (Gritti et al., 2001). . Briefly, the fetus was removed at the expense of a timed pregnant mouse. Under aseptic conditions, the brain was removed and the forebrain was excised. The tissue was separated by grinding, centrifuged at 75 g for 10 minutes to precipitate, and cells were seeded in an NSA medium containing growth factors in an uncoated tissue culture flask. Neurospheres were ground and seeded as single cells for passage.
As described by Shingo et al. (2001) about erythropoietin (EPO), asymmetric division (one stem cell, one neuronal progenitor cell or neuroblast) from neural stem cell symmetric division (two stem cells, both into stem cells) To determine if NRP can induce a shift of cells within the neurosphere to (5)), the neurospheres are ground and separated and placed in a 6-well plate in NSA medium supplemented with: Seeded for expansion at a density of 200,000 cells / well: EGF only; EGF + various concentrations of NRP; or EGF + 10 U / mL EPO. Every other day, half of the culture broth was replaced with a broth containing fresh NRP compound. Seven days later, neurosphere cells were plated. In the presence of EGF, the cell retains symmetrical division. With the removal of EGF, the cell begins to differentiate (asymmetric cell division). Subsequently, cells were subjected to a differentiation assay as described below except that bFGF was not added to the culture for the first 24 hours and was differentiated for 7 days in a control culture without NRP or other compounds. . In cells that were not exposed to NRP at all, the number of neuroblasts was less than in cells exposed to NRP or EPO. Therefore, NRP can enhance the differentiation of undifferentiated stem cells into neuroblasts.
Differentiation assay :
When full size was reached, the neurospheres were ground and separated and plated at a density of 200,000 cells per well on laminin-coated 13 mm diameter coverslips placed in Nunc 24-well plates. The culture medium of the plate culture was a 1: 1 mixture of DMEM / F12 supplemented with N2 and Neurobasal supplemented with B27 and 2 mM glutamine. The culture broth contained 2 ng / mL bFGF to enhance survival after plate culture. After 24 hours, the culture medium was replaced with a neuronal differentiation promoting medium (DMEM / F12 supplemented with N2 and Neurobasal 1: 3 mixture supplemented with B27 and 2 mM glutamine), and the test compound or IGF-1 except for the control. Was added at the same time. The culture medium containing the new compound was changed every other day. Seven days after the plate culture, the differentiated cells were incubated with a differentiation culture medium containing 100 ng / mL Syto21 for 20 minutes to label the nuclei of living cells. They were subsequently fixed with 4% paraformamide and immunostained with mouse anti-βIII tubulin antibody and goat anti-mouse Cy3-conjugated secondary antibody.
Quantification of neuronal differentiation and axonal outgrowth :
After staining, the cover slip was removed from the tissue culture plate and mounted on the cover slip using Immunofluore fluorescent mounting media. To analyze the percentage of neurons to total cells, Zeiss Axiophoto microscope equipped with Axoivision software was used, and any two fields per well at 20x magnification, red (tubulin stained neurons) and green (Syto21 stained nuclei) Investigated with fluorescent channel. The neurons and nuclei per field were counted and the percentage of neurons relative to the total cell number was determined. Results are shown in FIGS. 5A-5H.

Result :
When normalizing neuronal cell numbers within a differentiation assay over a wide concentration range within a differentiation assay, NRP-7 segment SW is a progeny of neuronal progeny cells generated from NSCs plated on laminin in differentiation media. Increased percentage. At a concentration of 10 pM, up to a 2-fold increase was observed in βIII-tubulin-positive neurons (FIG. 5E), while the greatest increasing activity in full-length axon growth was in the higher nanomolar concentration range (FIG. 5A). IGF-1 was used as a positive control for promoting axon outgrowth (Ishii et al., 1993) and was as effective as NRP, but was less potent when used at lower concentrations (Figure 5A). When rats were given 21mer NRP-9 with BrdU for 24 hours during NSC differentiation, an increase in proliferation rate could be observed (Figure 5H), indicating that the percentage increase in neurons in the assay was Increase the likelihood of at least in part due to proliferation of neuronal progenitor cells.
NRP-7SW and NRP-2KS had activity similar to that of IGF-1 and promoted axonal outgrowth of differentiated neural progenitor cells, but had much higher ability than IGF-1 ( Figures 5A and 5B).
Quantification of these data for NRP-2KS is shown by way of example in FIG. 5C (vehicle treatment) and FIG. 5D (NRP-2KS treatment).
When normalizing neuronal cell numbers within the differentiation assay over a wide concentration range, NRP-2KS gives a percentage of neuronal progeny that arise from NSCs plated on laminin in differentiation medium. Increased. At a concentration of 10 nM, up to a 2-fold increase was observed in βIII-tubulin-positive neurons (FIG. 5F). When NRP-2KS is administered for 24 hours with BrdU during NSC differentiation, an increase in proliferation rate can be observed (FIG. 5G), indicating that the increase in the percentage of neurons in the assay is at least partially Increases the likelihood that it results from proliferation of neuronal progenitor cells.

Example 5: Use of NRP to Promote Repopulation of Neural Populations In Vivo In view of the findings disclosed herein, we have found that NRP promotes population reformation of neural tissue in animals. Perform in vivo experiments to determine if you can. OEG cells were evaluated as a cell source for population remodeling of neural tissue following injury (injuries such as spinal cord injury). NRP is added to the OEG cell culture and / or co-administered with OEG cells in spinal cord transplantation procedures. A patient with a spinal cord injury is prepared for surgery at the site of injury and the spinal canal is accessed using methods known in the art to identify the area of injury. OEG cells and NRP are transplanted at the site of injury, optionally providing additional sources of NRP locally.

Example 6: Expression of NRP
Method :
Analysis of NRP gene expression: NRP gene expression in annotated mice was confirmed by RT-PCR, Northern blot and in situ hybridization.
In Situ Hybridization and Northern Blot: An 88mer oligonucleotide encompassing the unique coding region of mouse NRP (NRP-7 (SEQ ID NO: 35)) (upstream of the alkB homolog gene transcription start site) was transformed into pGEM7Zf (-) (Promega, Madison, WI, USA) was cloned between the BamHI and EcoRI sites. Complementary synthetic oligonucleotides also given appropriate 5 'overhangs (underlined); mfsNRP.S88, sense strand (Invitrogen) (shown below: 5' A ATT CGGAATGAATCGAAACCCTGGAGTCGTGACCCCGGAAGAACCTGCCAGAGCCGGAATTTCGAGTTCTGCTTCCGGGCCAAACTG (SEQ ID NO: 41) mfsNRP.AS88, antisense strand (Invitrogen) (shown below: 5 ' GATC CAGTTTGGCCCGGAAGCAGAACTCGAAATTCCGGCTCGGCAGGTTCTTCCGGGGTCACGACTCCAGGGTTTCGATTCATTCCG (SEQ ID NO: 42)) was further denatured, and further 100 mM NaCl, 0.1 mM EDTA (Sigma EP), 20 mM EDTA (Sigma) In 9), the oligonucleotides were heated at 95 ° C. for 2 minutes at 48 μM for 2 minutes, cooled to 60 ° C. at 1 ° C./minute, and maintained at 60 ° C. for an additional 1.5 hours. 1 μL of annealed 88mer and 300 ng Qiaex were extracted (Qiagen), gel purified, BamHI (Roche Diagnostics Ltd., Auckland, New Zealand) EcoRI (New England Biolabs Inc.) double digested pGEM7Zf (−) Ligated with 0.2 units of T4 ligase (Epicentre, Madison, WI, USA) for 19 hours at 4 ° C. Clones prepared with competent DH10B (Invitrogen) were screened for the presence of ClaI (Roche) resistant plasmid and 88mer excision upon EcoRI / BamHI double digestion. Templates for the synthesis of sense and antisense RNA probes were prepared by complete digestion of 10 μg of DNA (prepared using JetStar Maxi Kit (Genomed)) using 20 U BamHI or EcoRI, respectively. The template was gel purified using the Concert Rapid Gel Extraction System (Invvitrogen).
Riboprobes were transcribed and Northern blots were performed using the DIG Northern Starter Kit (Roche). 10 μg of total RNA from perinatal astrocytes or HEK cells transfected with pUSE-myc-mNRP was isolated on a 1.2% formaldehyde RNA gel with a 0.16-1.77 kb RNA ladder and positively charged in SSC Transfer to nylon membrane (Roche). The marker lane was cut out and stained with methylene blue. DIG-labeled NRP probe was hybridized overnight at 52 ° C. After stringent washing and incubation with DIG antibody, the signal was detected by CDP ^* luminescence using a bioimaging system (UVP). Whole brains were removed from E15 mice, fixed with 4% PFA for 3 hours, protected against freezing overnight with 20% sucrose, embedded in Tissuetek OCT medium and stored at -80 ° C . The cryoprotected brain was cut into 14 μm sections on a PLL-coated slide, treated with 8 μg / mal protease K for 8 minutes, post-stained with 4% PFA for 5 minutes, and further DIG-labeled NRP probe (88mer) at 45 ° C. Probe sequence) (1: 100 dilution) and hybridized overnight with sense control. After labeling with an anti-DIG antibody and developing a signal with NBT / BCIP, the sections were double-labeled with nestin / GFAP / b-tubulin and visualized with a fluorescent secondary antibody.

Result :
Using PCR, NRP messages were detected in various regions of the brain of E15 mouse fetuses, but interestingly, the expression levels were much higher in cultured mouse neural stem cells (FIG. 6A). Neural stem cell specific expression was further demonstrated by in situ hybridization to mouse NRP mRNA and double staining with stem / progenitor cell specific intermediate filament nestin (Lendahl et al., 1990) (FIGS. 6E-6H). To determine whether the NRP message (mRNA) is maintained in NSC-derived neurons or glial progeny cells, the expression level in undifferentiated NSCs is differentiated into astrocytes by CNTF and into neurons. Comparison with differentiated NSCs. NRP expression levels increased significantly only in CNTF-treated cells, indicating that the astrocytic lineage is the main supply of secreted proteins (Figure 6B). Comparison of expression levels in several tissues of E19 mice revealed that expression in non-neural tissues, except for the lung, was low and sometimes absent (FIG. 6C). Based on the relatively high level of expression in astrocytes, a northern blot with RNA from perinatal astrocyte cultures was attempted, which revealed two transcripts approximately 0.8 and 1.2 kb in size. . Since transcription begins at -114 and -509 upstream of the translation start, this is consistent with the expected full-length transcript (Figure 6D). In situ hybridization of E15 mouse fetal forebrain sections revealed strong signals in the marginal zone and subplate, complemented by weak labeling of the cortical plate (FIG. 6I). Mouse NRP mRNA showed co-localization with nestin immunoreactivity. At this stage, no co-labeling of GFAP or MAP2 was observed (data not shown). This staining pattern shows expression in radial glia-like cells, which have undergone transformation to immature astrocytes (Steindler & Laywell, 2003).

Example 7: Comparison of neuroprotection of cerebellar microexplant II-NRP-2 segment SW (SEQ ID NO: 24 and SDF-1)
Method :
Cerebellar microexplants were prepared as described in Example 9 with the addition of SDF-1 in addition to glutamate / 3-NP and NRP.
Result :
The damage caused by treatment of unprotected cerebellar microexplants with 0.5 mM 3-NP / glutamate for 48 hours ranged from 75-92% cell death. FIG. 7A shows that NRP-7SW provided very significant neuroprotection over a concentration range from 100 fM to 100 nM, with almost 50% recovery from injury at 100 pM. In comparison, 10 nM human SDF-1 conferred less than 30% neuroprotection and showed narrow dose range efficacy (FIG. 7B).
The 16mer cachexia fragment showed lower efficacy and had a 10-fold lower potency for neuronal protection and neuronal proliferation induction (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
Methods for contact migration assays :
The contact migration assay was performed using rat NRP-9SD (SEQ ID NO: 34) (4 nM) and mouse NRP-7SW (SEQ ID NO: 24) (0.4 nM) as attractants in Boyden chambers. Performed as described.
Thalamocortical System-Cortical Culture Method The occipital cortex and the thalamus from the newborn (P0) of Long Evans rats were excised according to Paxinos ³⁷ P0 atlas. The occipital cortex is coronal and the thalamic dorsal area is sliced 350 μm thick with a tissue shopper (McIlwain) from the front, and these are immediately supplemented with 0.65% D-glucose (Merck) Gay's Balanced Salt Solution (GBSS)) and allowed to recover at 7 ° C. for 1 hour. Thalamic sections with vertical orientation were sorted under a stereo microscope and placed on a coverslip together at least 3 mm away from the cortical tissue. In this case, the thalamus is placed in the direction where the bridle nucleus faces the cortical layer VI. These sections were adhered to the coverslips in plasma clots using 10 μL of chicken plasma (Cocalico) coagulated with 10 μL thrombin (25 U / mL, ICN). Place the coverslip in a roller tube (Nunc) and add 0.75 semi-artificial medium (2/4 reference medium eagle, 1/4 Hanks balanced salt solution, 1/4 inactivated horse serum, 2 mM L-glutamine and 0.65% D-glucose) was fed. Cultures were maintained in vitro in a roller tube incubator at 36 ° C. for up to 20 days (DIV), and the culture medium containing rat cachexia-related NRP was changed every 3 days.
Rat fetal forebrain OTCs were prepared as described. Briefly, a pregnant rat in pregnancy E17 stage was sacrificed and a fetus was quickly removed. Millicell PICM ORG50 membrane (Millipore) in a 3 cm Petri dish (containing 1 mL DMEM / F12, 6.5 mg / mL glucose, 0.1 mM glutamine and 10% FCS) cut into 400 μm thick coronal sections of the forebrain Left on top. Thereafter, the culture solution was exchanged with 1 mL of Neurobasal / B27, 6.5 mg / mL glucose, and 0.1 mM glutamine. 10 μL of PBS, NRP or SDF-1 is added to the above at the indicated concentration, and even smaller DiI (1,1′-Dioctodecyl-3,3,3 ′, 3-tetramethylindocarbocyanine) of uniform size The crystals were placed on the medial ganglion protuberance under a stereomicroscope (n = 10-15). After incubation for 24 hours, the total number or ganglion bumps and neurons dispersed in the cortex were counted under an inverted microscope.

Result :
NRP-9SD (SEQ ID NO: 34) (4 nM) and mouse NRP-7SW (SEQ ID NO: 24) (0.4 nM) showed substantial chemoattractant activity of NRP against mouse neural stem cells. Approximately four times the number of cells under control conditions were attracted by NRP-9SD, but less than one-tenth the amount of NRP-7SW still attracted more than twice NSC to the bottom of the culture dish. In comparison, 100 nM SDF-1 coating attracted a similar number of cells as with 0.4 nM mouse NRP-7SW (FIG. 36A). Their representative morphological phenotype is shown in FIG. 8B.
In an OTC assay using sagittal fetal brain slices containing ganglion bumps and cortical primordia, NRP-7SW administered to OTC enhanced the number of migrating neuronal precursor cells by a factor of 5 (Figure 8C). . Human SDF-1 induces a similar number of cells to migrate from the medial ganglion protuberance to the cortical primordia, but as in the case of the catalytic chemotaxis migration assay, mature human SDF-1 with a length of 63 amino acids Is much less capable than NRP.

Example 9: Expression and activity assay of recombinant NRP
Preparation of HEK293-expressing mfsNRP :
HEK293 cells were transfected with mNRP fusion protein expression constructs tagged at the N-terminus and C-terminus (pUSE-Flag-mNRP2 and pUSE-myc-mNRP11, respectively) with 25 kDa polyethyleneimine (Boussif et al. , 1995). The NRP sequence used was SEQ ID NO: 35. Parallel transfection with pEGFP-N1 (Clontech) was also performed. Briefly, each construct was combined with DNA phosphate at 10 ng / μL DNA in 5% glucose at a ratio of PEI to 6, 9, and 12 to 1 PEI. 70% confluent HEK239 cells in 2 mL growth medium (DMEM supplemented with 10% fetal bovine serum) in 36 mm wells were transfected by incubating with 200 μL per well of transfection mixture for 4.5 hours. This was followed by the addition of 2 mL growth medium containing 2 × antibiotic / anti-mycoplasma mix to each well. The next day, cultures were split 1:15 into growth media (containing 800 μg / mL geneticin) using 1 mL / well trypsin-EDTA. Sorting was maintained for 17 days by changing the culture medium twice a week. When confluence was reached, sorting was continued on the portion of cells split 1:10 and the rest frozen in a growth medium containing 10% DMSO. Furthermore, a 1: 2 split was prepared on day 13 from geneticin selection at 800 μg / mL. Stable transfectants were collected for detection of expressed tagged proteins.
The expression of Myc-NRP in HEK293 cells was confirmed by Western blot :
Western blot was performed to confirm the expression of Myc-NRP in HEK293 cells. Cell lysates were solubilized under denaturing conditions and proteins were separated using SDS polyacrylamide electrophoresis (PAGE). The expressed recombinant mouse Myc-NRP was detected by anti-Myc monoclonal antibody.

Result :
Generation of full-length recombinant murine NRP under the control of the c-myc promoter transfected into HEK cells reveals the expected 0.8 kb transcript when using an antisense 88 bp NRP cRNA probe (Figure 9A). The 16.5 kDa recombinant protein product expressed by Myc-NRP-HEK cells migrated to a 20 kDa marker size on a Remli SDS gel (FIG. 9B).
The measured biological activity of the complete NRP gene recombinant expression product encoded by the NRP gene was the same as that of the NRP synthetic peptides tested. In co-culture assays with cerebellar microexplants, Myc-NRP-HEK cells confer substantial neuroprotection when seeded at various cell concentrations, with a Myc-NRP-HEK cell count of 5000 and a recovery value of It was 51% of MAP-2 positive neurons (Fig. 9C). Control Myc-HEK containing only empty vector showed no recovery of MAP-2 positive cells after oxidative / excitotoxic stress. The added HEK cells showed a bell-shaped dose response curve for neuroprotection.
Myc-NRP-HEK cells showed chemoattractant neuron migration-inducing activity when tested in a catalytic chemotaxis migration assay using mouse NSCs. When Myc-NRP-HEK cells expressing mouse NRP are seeded at the bottom of the Boyden chamber and mouse NCS is seeded on the insert, the neuronal cell population showing neurite outgrowth detected 24 hours later is the control Myc- There was a 2-fold increase compared to the same assay using HEK cells (Figure 9D).
Example 10: Possible mechanism of action of NRP :
Recent data suggests that SDF-1 (CXCL12) and its receptor CXCR4 may have similar effects in the immune and nervous systems. That is, they can regulate cell migration, neuronal and lymphocyte proliferation, plasticity (neurite outgrowth and differentiation) and survival (Vlahakis et al., 2002 and Lazarini et al., 2003). Similar dependence on CXCL12 may support cooperative homeostasis interactions, but as a result of CXCR4 receptor binding inherent vulnerabilities to inflammatory processes such as HIV infection and subsequent neuropathy (Keswani et al., 2003). HIV and related viruses require a co-receptor in addition to the lymphocyte receptor CD-4 to infect target cells. One co-receptor used for HIV is the G-protein coupled chemokine receptor CXCR4. The interaction between SDF-1 and CXCR4 can block the entry of HIV into CD4-lymphocytes. Furthermore, it is known that inhibition of cancer cell migration / invasion by antagonizing the CXCR4-receptor can prevent cancer metastasis in animal models (Rubin et al., 2003; Liang et al. al., 2004).
To date, SDF-1 is the only known ligand that binds to the CXCR4 receptor. We here provide some evidence that NRP can be a new class of ligands for the CXCR4 receptor and that biological activities (eg chemoattraction and neuronal survival) can be exerted by activation of the CXCR4 receptor To do. Thus, NRP can be an agonist for the CXCR4 receptor, while not excluding the possibility that there may be an antagonistic effect of NRP on the CXCR4 receptor. Similar observations were obtained for SDF-1. That is, single amino acid substitutions can antagonize SDF-1 action on CXCR4 (Tudan et al., WO0185196). However, it will be appreciated that this is not the only possible mechanism of action of NRP, and that other mechanisms may explain this observation described herein.

Method :
Cerebellar microexplants were prepared as described in Example 9 with the addition of SDF-1, wortmannin and PD98059 (both Calbiochem) in addition to glutamine / 3-NP and NRP.
Effect of CXCR4 receptor neutralizing antibody on NRP survival promotion and migration inducing activity
By applying 1 μg / mL of neutralizing antibody prepared against CXCR4 receptor (Fuzin receptor) for 72 hours, neuronal survival and neuronal migration patterns in cerebellar microexplants treated with NRP were significantly reduced. At an antibody concentration of 1 μg / mL, NRP-9 SD (SEQ ID NO: 34) lost its neuroprotective and / or migration promoting effect (FIG. 10A). Similar inhibition was observed for human NRP-2KS (SEQ ID NO: 23) (FIG. 10B) or mouse NRP-7SW (SEQ ID NO: 24) (data not shown).
Effect of CXCR4 receptor neutralizing antibody on chemotaxis of NRP Chemoattractant effect of 10 ng / mL NRP coated on culture dish is to pre-incubate neuronal stem cell line MEB-5 with neutralizing antibody against CXCR4 for 1.5 hours Blocked by. Very few cells migrated compared to the migration observed with the NRP-9SD (SEQ ID NO: 34) peptide alone (FIG. 10C).
In order to elucidate the signaling pathways utilized by NRP to exert neuroprotective and / or migratory effects that the action of NRP may be mediated by ERK1 / 2 and Akt phosphorylation , we investigated the MEK inhibitor PD98509 and phosphatidylinositol The 3-kinase (PI-3K) inhibitor wortmannin was used to inhibit MAPK or Akt phosphorylation.
To find out the role of PI-3K (Akt phosphorylation), an upstream regulator of MAPK and akt activity, in the mechanism of NRP-mediated neuroprotection and chemoattraction, we used cerebellar microexplants and catalytic chemotaxis migration assays. The MAPK kinase (MEK) inhibitor PD98509 was tested for efficacy in. Injury treatment with or without vehicle and PD98509 showed only mild toxicity with the inhibitor, while co-treatment with inhibitor and NRP-9SD (SEQ ID NO: 34) lost NRP neuroprotective activity (Figure 11A). Inhibition of Akt phosphorylation by wortmannin (100 nM) had no significant effect after vehicle and NRP-free injury treatment, but similar to inhibition of ERK1 / 2 phosphorylation, neuroprotection produced by NRP Lost (Figure 11B). In the catalytic chemotaxis migration assay, almost 3-fold increase in migratory NSC was almost completely blocked by PD98509 (0.1 pM to 100 pM) and had no significant effect on basal migration in BSA-coated wells (Figure 11C). ). In contrast to neuroprotection, chemoattractant migration activity of NRP was not suppressed by inhibition of PI-3K by wortmannin (data not shown). To eliminate the possibility that the decrease in the number of cells that migrated to the bottom compartment was due to impaired neuronal survival, the inserts were stained for live cells with Syto2 and the top surface was analyzed. No significant differences were found in viable cells.

Example 11: NRP efficacy in vivo
Materials and methods :
To test the effectiveness of NRP in vivo, experiments were conducted in rats exposed to hypoxic ischemic injury (HI). Adult rats (50 days old, Wistar rats, 250-300 g, male) were used. Modified Levine model preparation and experimental methods were used (Rice et al., 1981, Ann. Neurol. 9: 131-141; Guan et al., 1993, Cereb. Blood Flow Metab. 13 (4) : 609-16). Briefly, these methods consist of HI injury induced by unilateral carotid artery ligation in an animal with a lateral cannula implanted cannula followed by inhaled asphyxia. By stereotaxic surgery under halothane anesthesia, a guide cannula was placed 1.5 mm to the right of the midline on the top of the dura mater and 7.5 mm forward of the interaural zero plane. Two days after cannulation, the right carotid artery was double ligated. After a 1 hour recovery time from anesthesia, each rat was placed in an incubator with controlled humidity (90 ± 5%) and temperature (31 ° C ± 0.5 ° C) for an additional hour followed by each rat being hypoxic (6 % Oxygen) for 10 minutes. Animals were maintained in an incubator for an additional 2 hours prior to treatment.
After hypoxic ischemic injury, rats were treated with 5 nM (n = 12), 50 nM (n = 12) or 500 nM (n = 12) of NRP-4 segment PQ (PGRAEAGGQ; SEQ ID NO: 43) dissolved in saline. Or intraventricular (icv) administration of vehicle (n = 10) (normal saline).
Histological examination was performed on rats 5 days after hypoxic ischemia. Rats were killed with excess pentobarbital and perfused with normal saline followed by 10% formalin via the heart. Brains were kept in the same fixative for a minimum of 2 days prior to treatment and standard paraffin embedding was used.
A coronal section 8 μm thick was cut from the striatum, cerebral cortex and hippocampus and stained with thionine and acidic fuchsin. The severity of tissue damage was scored by CA1-2, CA3, CA4 and dentate gyrus of the striatum, cortex and hippocampus. Tissue injury was identified as neuronal loss (acidophilic (red) cytoplasm and condensed nuclei), extensive necrosis and cellular response. Tissue injury was scored using the following scoring system: 0: tissue did not show tissue injury; 1: <5% tissue was injured; 2: <50% tissue was injured; 3:> 50% of the tissue was injured; and 4:> 95% of the tissue was injured.
Results and conclusions :
The results of this experiment are shown in FIG. 100% in all analyzed brains 5 days after injury by 9-mer fragment of human chromosome 15, NRP-4 segment PQ (PGRAEAGGQ-NH ₂ ; SEQ ID NO: 43; 9 ng / icv 2 hours after hypoxia) Of neuroprotection.

Example 12: Growth promotion of olfactory bulb sheath glia ("OEG") cells
To determine if NRP could be useful for neural tissue remyelination, we performed experiments with human ensheating glial (OEG) cells. OEG cells have been evaluated as a supply population of cells for repopulation of damaged tissue (eg, spinal tissue). OEG cells were obtained from scraping specimens of human nasal mucosa using methods known in the art. Cells were grown in culture under controlled conditions and in the presence of NRP-7SW (SEQ ID NO: 24).
Under controlled conditions, the normal growth rate of OEG cells is about 25% within 72 hours. We have found that in the presence of NRP-7SW (SEQ ID NO: 24), OEG cells proliferate at a rate of approximately 50% over 72 hours. These results indicate that NRP can enhance the proliferation of OEG cells, thereby being useful in nerve cell transplantation methods, promoting cell proliferation and restoring nerve function to the damaged tissue (FIG. 13). ).

Example 13: Neuroprotective activity of NRP-5 segment RG peptide analog
To determine whether an alteration in the amino acid sequence of an NRP-5 segment RG-related peptide produces a peptide having an activity different from that of NRP-5 segment RG (SEQ ID NO: 30), we Synthetic peptides with substitutions were made. We tested the substituted NRP-5RG peptide in the cerebellar microexplant assay disclosed herein.
Amino acid substitutions within the first five amino acids of the N-terminus of the amidated peptide having the amino acid sequence REGRRDAPGRAGG-NH ₂ (SEQ ID NO: 30) result in the sequences REAAADAPGRAGG-NH ₂ (SEQ ID NO: 44) and AAARRDAPGRAGG-NH ₂ A peptide having (SEQ ID NO: 45) was prepared.
Amino acid substitution in the PGR domain resulted in a peptide having the amino acid sequence REGRRDAAAAAGG-NH ₂ (SEQ ID NO: 47), and substitution of the C-terminal GG domain resulted in a peptide having the amino acid sequence REGRRDAPGRAAA-NH ₂ (SEQ ID NO: 46) . Each of the above substituted peptides was tested and the results compared with the results obtained using NRP-5 segment RG (SEQ ID NO: 30; “NRP-5RG”).
FIG. 14A shows that SEQ ID NO: 44 had neuroprotective activity with a maximum effect observed at a concentration of about 1 pM. The bar on the right (labeled “standard 13mer”) indicates NRP-5RG (SEQ ID NO: 30). Substitution of amino acids 3-5 of NRP-5RG did not significantly change the activity profile of the peptide (FIG. 42A).
FIG. 14B shows that SEQ ID NO: 45 has neuroprotective activity, with a maximum effect observed at about 1 pM. The bar on the right (labeled “prototype sequence”) indicates NRP-5RG (SEQ ID NO: 30). The substitution at positions 1-3 resulting in AAARRDAPGRAGG-NH ₂ (SEQ ID NO: 45) resulted in a higher neuroprotective activity than the original sequence NRP-5RG. This result may be due to the stability profile of this analog higher than NRP-5RG. However, other hypotheses can explain this observation.
FIG. 42C shows that SEQ ID NO: 46 is more than NRP-5RP (SEQ ID NO: 30; right bar; “standard 13mer”) or SEQ ID NO: 44 or SEQ ID NO: 45 (FIGS. 14A and 14B, respectively). It has a low effect. In fact, a statistically significant effect was only observed at a concentration of 100 nM. Prototype sequence (SEQ ID NO: 30) produced a 20% higher neuroprotection, SEQ ID NO: having 10 ⁷ times 10 ⁶ than 46 high potential.
FIG. 14D shows that SEQ ID NO: 47 has some neuroprotective effect at certain concentrations (eg, 0.1 pM, 10 pM and 100 nM), but at other concentrations NRP-5RG (SEQ ID NO: 30; labeled “standard”) The right bar) was not active compared to that observed.
We concluded from these experiments that the PGR and C-terminal GG domains are useful for maintaining the activity of NRP-5RG (SEQ ID NO: 30). We also concluded that the amidated C-terminus of NRP is not sufficient to produce a neuroprotective effect. This is because C-terminal amidated NRP (SEQ ID NO: 46) had little, if any, neuroprotective effect at most concentrations. Furthermore, changes within the NRP affected activity even if none of the internal amino acids had a C-terminal amide group.

Example 14: Growth induction and chemoattractant activity of NRP-5RG In another set of experiments, we determined the effect of NRP-5RG on the induction of neuronal proliferation in fetal cerebellar microexplants described herein. did. FIG. 15 shows that NRP-5RG has growth inducing activity, with maximum activity observed at a concentration of 100 pM. Some effects were observed at 1 mP and even at a concentration of 0.1 pM, but these effects were not statistically significant.
In yet another experiment, we examined the chemoattractant effects of NRP-5RG in the catalytic chemotaxis migration assay described herein. FIG. 16 shows that NRP-5RG is chemoattractant and has a 42.1% stronger effect compared to cells exposed to control (containing BSA) cultures.

Example 15: In this experimental series where NRP protected neurons in response to oxidative stress , we show that NRP-4 segment PQ (“NRP-4PQ”) protects cerebellar microexplants from oxidative damage I investigated whether it was possible. We exposed the cells to vehicle, 0.1 mM hydrogen peroxide, or various concentrations of NRP-4PQ in the vehicle. FIG. 17 shows the neuroprotective effect of NRP-4PQ 48 hours after oxidative stress on 0.1 mM hydrogen peroxide. The neuroprotection associated with NRP-4PQ treatment exceeded even that of control explants that did not receive peroxide. The mechanism of this enhanced efficacy of neurite growth is not clear, but it may alleviate the stress associated with creating or maintaining the microexplant itself.
These findings are related to many conditions in which oxidative stress plays a role. For example, oxidative stress is associated with all chronic CNS injury and disease. Suppression of oxidative stress-mediated neurotoxicity by NRP action can be very beneficial for many CNS injuries or diseases.

Example 16: Neuroprotection mediated by phosphorylated NRP-7SW We performed a series of experiments where phosphorylation of the N-terminal amino acid serine is neuroprotective of NRP-7 segment SW ("NRP-7SW") It was decided whether to change the action. NRP-7 ^P SW is generated by phosphorylation of NRP-7SW. Because of the high probability (0.9) that serine is phosphorylated under in vivo conditions ( NetPhos 2.0 Server— Technical University of Denmark), we hypothesized the role of N-terminal serine. The Cerebellar Microexplants to damage in 0.5mM of 3-NP / glutamate, it was treated with NRP-7 ^P SW.
18, NRP-7 ^P SW shows the experimental results showing that have neuroprotective effects. A significant effect was observed at concentrations as low as 0.1 pM, which showed higher neuroprotective activity (44.2% neuroprotection) than the other concentrations tested. Since the lowest concentration used (0.1 pM) showed considerable neuroprotection, even lower concentrations would show neuroprotection.

Example 17: Enhanced NSC proliferation after induction of differentiation by NRP-9SD
E15 mouse NSC P10 cells were seeded on laminin and cultured for 3 days in the presence of NRP-9SD (SEQ ID NO: 34), and in the presence of BrdU for the last 48 hours of the culture period. For each condition, 4 fields were counted in 2 separate wells to determine the number of proliferating cells.
Result :
NRP-9SD increased the proliferation rate of differentiating mouse NSC precursor cells (FIG. 19). This biological activity was observed over a wide dose range from 100 fM to 1 nM.

Example 18: Expression of NRP II
In situ hybridization :
Whole brains were removed from E15 and E17 mice, fixed with 4% paraformaldehyde (PFA) for 3 hours, cryopreserved overnight with 20% sucrose, and embedded in Tissuetek OCT (Sakura finetek)- Stored at 80 ° C. This cryoprotected brain was cut into 14 μm-thick sections, placed on a PLL-coated slide, treated with 8 μg / mal proteinase K for 8 minutes, fixed with 4% PFA for 5 minutes, and further DIG-labeled NRP sense. And an antisense probe at 45 ° C. overnight (88mer probe sequence described in Example 13; sense strand is SEQ ID NO: 41, antisense strand is SEQ ID NO: 42; diluted 1: 100). After labeling with anti-DIG antibody and color development with NBT / BCIP, sections were double labeled with 1: 100 dilution of nestin / GFAP / βIII-tubulin and vimentin (abcam, mouse monoclonal [RV202], ab8978-1) Visualization was performed using a secondary fluorescent antibody. The sense control remained negative.
Result :
Figures 20A and 20B show fluorescence micrographs of the above-described animal brains. 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 (FIGS. 20A and 20B). This points to an important function of neural stem cells. This is because radial glia have been shown to generate neurons in many brain regions during development (TE Anthony, C. Klein, G. Fishell, N. Heinz, 2004 ”Radial glial cells serve as neuronal progenitors in all regions of the central nervous system. ”Neuron, 41: 881-890), the astrocytes in the adult subventricular zone have been shown to be neural stem cells (F. Doetsch, I. Caile, DA Lim, JM Garcia- Verdugo, A. Alvarez-Buylla, 1999 “Subventricular zone astrocytes are adult stem cells”, Cell 97: 703-706 ”).
From these results we concluded that NRP mRNA is produced in the mouse brain.

Example 19: Rearrangement of SEQ ID NO: 8 and SEQ ID NO: 9
On August 20, 2004, a report of an updated sequence corresponding to SEQ ID NO: 8 and SEQ ID NO: 9 was published (NBCI, NT_026446). Based on the newly identified sequence, we annotated that the nucleotide NRP sequence consists of 225 nucleotides that encode a peptide with 75 amino acids. According to the previously annotated sequence on chromosome 15 encoding NRP (SEQ ID NO: 8), the following was changed in said sequence: position 168 changed from G to C, otherwise exons 1-4 No change (including biologically active sequence of NRP). Newly annotated exon 5 starts from 179th. Exon 6 starts from 222nd.

Write the entire amino acid sequence in one-letter code:
> Humchrom15NRP exon 1 2 3 4 5 6
MAVVLLAPFGDISQEITKVGTGTPGRAEAGGQVSPCLAASCSQAYGAILAHCNLCLPGSSDLPASASQSARLQVD
SEQ ID NO: 49

Example 20: Treatment of stroke The patient is showing signs of stroke. A diagnosis of stroke is made and the physician then administers the NRP compound to the patient intravenously or directly into the ventricle or directly to the affected part of the patient's brain. The NRP compound is a peptide or protein as disclosed herein and is administered in a pharmaceutically acceptable form (including vehicles, buffers and stabilizers if desired). Treatment with NRP reduces neurodegeneration associated with stroke and further delays, stops or at least partially reverses the expected worsening of symptoms.

Example 21: Prophylactic use of NRP A patient is diagnosed with cardiovascular dysfunction and coronary artery bypass (CABG) surgery is indicated. CABG surgery is associated with reduced brain perfusion, which can lead to hypoxic or ischemic brain injury. In order to reduce the adverse effects of such hypoxia and ischemia, patients are pretreated with NRP compounds. The NRP compound is administered to the patient in a pharmaceutically acceptable form (including vehicle and / or stabilizer if desired). Routes of administration include intravenous, intracerebral or ventricular administration. Multi-route administration can be used if desired. Pretreatment of patients undergoing CABG surgery reduces neurodegeneration associated with CABG surgery, and patients experience postoperative neurological loss compared to patients undergoing CABG surgery without NRP pretreatment. Decrease.
The invention has been described with reference to specific embodiments thereof. Those skilled in the art will be able to develop other embodiments incorporating the disclosure and teachings of the present application without undue experimentation. All of these embodiments are considered part of this invention. All references cited herein are hereby incorporated by reference.

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Industrial Applicability Embodiments of the present invention include genes and peptides for nerve regeneration peptides (NRP) useful for the manufacture of compositions used for the treatment of conditions including neurodegeneration or neuronal cell death. Treatment with NRP increases neuronal survival, neuronal migration, neuronal differentiation, neurite outgrowth and / or neuronal proliferation. Conditions such as Alzheimer's disease, Parkinson's disease, hypoxia, stroke and coronary artery bypass surgery can be effectively treated by the compositions and methods of the present invention.

FIG. 1A shows the structure of the gene encoding NRP-7 (SEQ ID NO: 35) with an intron, along with the presence of the NRP gene as a 2 bp frameshift of a known gene encoding a DNA repair protein. The promoter region containing two CpG islands is highlighted (one CpG island is expected for NRP and the other downstream is expected for DNA repair protein). The position of the primer for obtaining the NRP-7 (SEQ ID NO: 35) gene product is indicated. FIG. 1B shows 72.2% homology between mouse NRP (NRP-7 long) and rat NRP (NRP-9) orthologs. Red numbers indicate corresponding homologies within biologically active NRP domains, while blue lines indicate putative N-glycosylation sites. FIG. 1C shows an alignment between human cachexia-related protein and 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%. The biologically active domain of cachexia-related protein is located immediately after the signal peptide at position 20, while the active domain of mouse NRP (NRP-7 long) starts at amino acid position 83. FIG. 1D shows that the overall homology between the mouse NRP fragment and the human trefoil protein Ps2 is 52.5%. Of the 15 amino acid residues that define the consensus sequence of the trefoil factor family (TFF), 9 amino acids are present in NRP, thereby confirming that the trefoil factor domain is present in NRP. FIG. 1E shows an alignment of mouse SDF-1α with mouse NRP (NRP-7 long) protein sequence. There is 32.6% moderate homology between both neuron chemoattractants. FIG. 6 shows survival induction by NRP-4 segment GQ (SEQ ID NO: 26) after excitotoxic / oxidative damage using 3-NP / glutamate. FIG. 6 shows proliferation induction by NRP-7 segment SW (SEQ ID NO: 24) in neurons damaged with 3-NP / glutamate. FIG. 6 shows a haptotactic migration assay by mouse NEB-5 cells using 10 ng / mL NRP-4GG (SEQ ID NO: 29) peptide for coating. 200,000 MEB-5 cells were used. The result of the migration assay by the primary mouse stem cell using 10 ng / mL NRP-4 GG (sequence number: 29) peptide for a coating is shown. 200,000 cells were seeded. The results of migration assays with mouse NEB-5 cells using 1 ng / mL NRP-2KS (SEQ ID NO: 23) peptide coating are shown. 200,000 cells were seeded. Figure 2 shows a migration assay with primary mouse stem cells using 10 ng / mL NRP-7SW (SEQ ID NO: 24) coating. 400,000 cells were seeded. FIG. 5 shows a migration assay with wild type PC-12 cells (200,000 cells / well) using a 50 ng / mL NRP-4GG (SEQ ID NO: 29) coating. Figure 2 shows a migration assay using wild type PC-12 cells and 1 ng / mL NRP-7SW (SEQ ID NO: 24) coating followed by Matrigel / PDL coating. 100,000 cells were seeded. FIGS. 5A and 5B are graphs showing the intermediate length of the longest five axons in each culture well of neural stem cells differentiated for 7 days in the presence of NRP. FIG. 5A shows the effect of NRP-7SW (SEQ ID NO: 24) and IGF-1. FIGS. 5A and 5B are graphs showing the intermediate length of the longest five axons in each culture well of neural stem cells differentiated for 7 days in the presence of NRP. FIG. 5B shows the effect of NRP-2KS (SEQ ID NO: 23), IGF-1 or differentiation culture medium alone. Significant differences from control cultures were observed as determined by 2-tailed Student's t-test. ^** p <0.01; ^*** p <0.001 5C and 5D show representative examples of the longest axon outgrowth in the case of differentiation medium alone. 5C and 5D show representative examples of the longest axon outgrowth in the case of differentiation medium alone. FIG. 5D shows longest axon growth in the presence of 10 nM NRP-7 SW (SEQ ID NO: 24), and FIG. 33D shows control conditions. FIGS. 5E and 5F are graphs showing the effect on the increase of NRP-7SW (SEQ ID NO: 24) and NRP-2KS (SEQ ID NO: 23) in NSC cells that have undergone neuronal differentiation. FIGS. 5E and 5F are graphs showing the effect on the increase of NRP-7SW (SEQ ID NO: 24) and NRP-2KS (SEQ ID NO: 23) in NSC cells that have undergone neuronal differentiation. It has been shown that nanomolar concentrations of NRP-2KS (SEQ ID NO: 23) promote the generation of neuronal precursors at the expense of pluripotent stem cells derived from mouse forebrain neural stem cells. FIG. 5 shows administration of NRP-9 segment SD (SEQ ID NO: 34) to undifferentiated NSCs prior to the start of a differentiation experiment scheme. Upregulation of BrdU positive cells occurs under NRP treatment, but there is no obvious change in the overall cell number. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6A shows that mouse NRP expression was detected in the cortex (Ctx), striatum (Str) and olfactory bulb (OB) of E15 mouse fetal brain tissue. However, the levels were much lower compared to mouse neural stem cells that were acutely isolated and immortalized. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6B shows that differentiation of neural stem cells into astrocytes by CNTF significantly increases NRP mRNA expression compared to neuronal differentiation by undifferentiated stem cells or BDNF. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6C is a gel showing analysis of various E19 mouse fetal tissues. The above indicates that mouse NRP expression is low or absent in non-neural tissues, except lung, compared to fetal cortex, astrocyte-differentiated NSCs and astrocyte cultures derived from the perinatal forebrain Is shown. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6D shows Northern blot hybridization with an 88 bp probe that does not overlap with the DNA repair protein sequence. This detects two separate mRNAs of approximately 0.8 and 1.2 kb in size from RNA derived from perinatal astrocyte cultures. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6E shows in situ hybridization with a mouse NRP antisense probe that labels NSCs. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6F shows the lack of specific signal in the sense control. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6G shows NRP mRNA expression in NSC. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6H shows that nestin is distributed at the same site as the NRP mRNA expression shown in FIG. 6G. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6I shows in situ hybridization of coronary NRP mRNA in E15 mouse forebrain sections, ventricular zone, cortical primordia (especially subplates and marginal zone), where the intensity of hybridization is reduced in the cortical plate. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6J shows nestin positive cells extending the entire length from the subplate to the marginal zone. 6A-6K show NRP gene expression in NSCs and in mouse fetal tissues. 6A-6C are photographs of gels showing that in multiplex PCR, the expression level of mouse NRP (upper band, 412 bp) is quasi-quantitative compared to β-actin expression (lower band, 260 bp). . FIG. 6K shows co-expression of mouse NRP message at the same site as nestin expression in FIG. 6J. FIG. 6L shows NT-2 cell NRP-2 (SEQ ID NO: 4) gene product expression in the absence of damage and 6 hours after damage caused by the mitochondrial toxin 3-nitropropionic acid. Gene expression was substantially reduced when cells were treated with 3-NP for more than 1 hour. 7A and 7B show neuronal survival and proliferation induction by NRP-7 segment SW. FIG. 7A shows the effect on neuroprotective activity of NRP-7SW (SEQ ID NO: 24) over a wide dose range from 0.1 pM to 100 nM. 7A and 7B show neuronal survival and proliferation induction by NRP-7 segment SW. FIG. 7B shows that SDF-1 had only a limited neuroprotective effect compared to NRP. 8A and 8B show the results of a neuron migration induction experiment using NRP. FIG. 8A shows that rat and mouse synthetic peptide-derived NRPs (NRP-9SD and NRP-7SW) exhibit chemoattractant properties in the attraction of neuronal stem cells (“NSC”) as shown in the catalytic chemotaxis migration assay. It is a graph which shows. The effectiveness of NRP-7SW was similar to that of SDF-1, but NRP showed higher potential. Although the scale of action of SDF-1 and NRP-7SW appears to be similar, the amount of SDF-1 (100 nM) is greater than the amount of NRP-7SW (0.2 nM). NRP-7SW is therefore about 500 times more powerful than SDF-1. 8A and 8B show the results of a neuron migration induction experiment using NRP. FIG. 8B is a micrograph of a brain OTC showing migration of neural progenitor cells derived from central ganglion ridges that migrate towards the cortical primordia. Figure 2 shows a graph of the quantification of an OTC assay showing the maximum effect of NRP-7SW at a concentration of 1 nM. On the other hand, the capacity of SDF-1 was lower than the above. Student's t-test was used for statistical analysis (* p <0.05, ** p <0.01, *** p <0.001; N = 6). 9A-9D show the results of expression and functional experiments of full-length mouse recombinant NRP-7 long (SEQ ID NO: 35). FIG. 9A shows that under the control of the cytomegalovirus (“CMV”) promoter, the 0.8 kb signal of the mouse NRP-7 long (SEQ ID NO: 35) gene was detected by HEK when detected by an NRP gene-specific 88 bp cRNA probe. A Northern blot showing very over-expression in cells is shown. 9A-9D show the results of expression and functional experiments of full-length mouse recombinant NRP-7 long (SEQ ID NO: 35). FIG. 9B is a Western blot showing that NRP-7 long (SEQ ID NO: 36) is expressed by HEK cells and migrates with a molecular weight of 20 kDa. 9A-9D show the results of expression and functional experiments of full-length mouse recombinant NRP-7 long (SEQ ID NO: 35). FIG. 9C is a graph showing that recombinant Myc-NRP-7 long (SEQ ID NO: 35) -HEK cells provide 51% neuroprotection against oxidative / excitotoxic damaged cerebellar microexplants. It is. 9A-9D show the results of expression and functional experiments of full-length mouse recombinant NRP-7 long (SEQ ID NO: 35). FIG. 9D shows that Myc-NRP-7 long (SEQ ID NO: 36) -HEK cells have chemoattractant activity to attract NSCs in a catalytic chemotaxis migration assay when seeded at the bottom of the culture dish. It is a graph. Compared to the Myc-HEK control experiment, more than twice as many cells were attracted from the insert to the bottom of the culture dish. Student t-test was applied for statistical analysis (*** p <0.001 for microexplants; N = 4, and *** p <0.001 for catalytic migration assay; N = 6). Figures 10A-10C show the results of experiments on P4-cerebellar explants using antibodies against CSCR4 and NRP. FIG. 10A is a graph showing inhibition of NRP-9SD (SEQ ID NO: 34) on neurite outgrowth. Figures 10A-10C show the results of experiments on P4-cerebellar explants using antibodies against CSCR4 and NRP. FIG. 10B shows inhibition of NRP-2KS (SEQ ID NO: 23) for neurite outgrowth. The chemoattractant effect of 10 ng / mL NRP coated on the culture dish was completely inhibited by preincubating the neuronal stem cell line MEB-5 with a neutralizing antibody against CXCR4. Figures 10A-10C show the results of experiments on P4-cerebellar explants using antibodies against CSCR4 and NRP. FIG. 10C shows that the antibody against CXCR4 reduced the chemoattractant effect of NRP-9SD compared to cells treated with NRP-9SD (SEQ ID NO: 34) alone. FIGS. 11A-11C show the results of experiments on the action of NRP with MAPK inhibitors, proposing possible signaling chain events that are centrally involved in the neuroprotective and migration-inducing activities of NRP. FIG. 11A is a graph showing that the MAPK (MEK) -inhibitor PD98509 completely inhibits the neuroprotective activity of rat NRP-9SD (SEQ ID NO: 34) over various NRP concentration ranges. FIGS. 11A-11C show the results of experiments on the action of NRP with MAPK inhibitors, proposing possible signaling chain events that are centrally involved in the neuroprotective and migration-inducing activities of NRP. FIG. 11B is a graph showing that, like PD98509, the PIK-3 inhibitor wortmannin inhibits the neuroprotective activity of NRP-9SD (SEQ ID NO: 34). FIGS. 11A-11C show the results of experiments on the action of NRP with MAPK inhibitors, proposing possible signaling chain events that are centrally involved in the neuroprotective and migration-inducing activities of NRP. FIG. 11C is a graph showing that the number of migratory NSCs induced by NRP-9SD (4 nM) is inhibited by 15 μM PD98509, while the basal migration level in BSA-coated wells is not significantly altered by MEK inhibitors. Yes (* p <0.05, ** p <0.01, *** p <0.001; N = 8). When administered ICV 2 hours after hypoxia, NRP-4 segment PQ (SEQ ID NO: 43) shows the results of an experiment that provided substantial neuroprotection in all of the brain areas analyzed 5 days after injury . NRP-5 segment RG (SEQ ID NO: 30) administered ICV 2 hours after hypoxia shows the results of an experiment that provided substantial neuroprotection in all of the brain areas analyzed 5 days after hypoxia . NRP-7 segment SW (SEQ ID NO: 24) is the result of an experiment showing that the growth of primary cultures of human adult olfactory bulbous glia (ensheating glia) is enhanced. Figures 14A-14D show experimental results for the prototype NRP-5RG sequence of the neuroprotective activity of four NRP-5 RG analogs. Figures 14A-14D show experimental results for the prototype NRP-5RG sequence of the neuroprotective activity of four NRP-5 RG analogs. Figures 14A-14D show experimental results for the prototype NRP-5RG sequence of the neuroprotective activity of four NRP-5 RG analogs. Figures 14A-14D show experimental results for the prototype NRP-5RG sequence of the neuroprotective activity of four NRP-5 RG analogs. The graph of the proliferation-inducing action of NRP-5 RG in fetal cerebellar cells is shown. It is a graph which shows the result of a chemotaxis migration assay using mouse | mouth NSC and NRP-5 RG as a chemoattractant. It is a graph which shows the result of the neuroprotective effect of NRP-4 PQ 48 hours after oxidative stress (0.1 mM hydrogen peroxide). It is a graph which shows the neuroprotective action of phosphorylated NRP-7 SW (NRP-7 ^P SW). It is a graph of the experiment which shows the effect | action of NRP-9SD with respect to a BrDU dyeing | staining nucleus. FIGS. 20A and 20B show photomicrographs of gene expression of NRP-7 (SEQ ID NO: 35) within the neuroepithelial stem cell-radial glia-astrocytic lineage. FIG. 20A shows the expression of mouse frameshift NRP transcript in the cerebral cortex of E17 mouse brain. Detected by in situ hybridization on frozen sections using a 88mer specific probe. FIGS. 20A and 20B show photomicrographs of gene expression of NRP-7 (SEQ ID NO: 35) within the neuroepithelial stem cell-radial glia-astrocytic lineage. FIG. 20B shows light microscopy using anti-vimentin antibody in the same section. A high degree of co-localization of the mouse frameshift message and the filament protein of the vimentin intermediate is shown.

Claims (5)

  Use of an NRP compound consisting of the amino acid sequence of SEQ ID NO: 30 in the manufacture of a medicament for promoting nerve regeneration by neuroprotection.   Nerve regeneration is at risk for neurodegeneration, cell death, or Parkinson's disease, Alzheimer's disease, hypoxia, ischemia, stroke, coronary artery bypass surgery, trauma, traumatic brain injury, Huntington's disease, spinal cord injury, infection, The use according to claim 1, for treating cells that have been degenerated by one or more of medical procedures selected from the group consisting of optic neuritis or surgery, chemotherapy and radiation therapy.   In vitro use of an NRP compound consisting of the amino acid sequence of SEQ ID NO: 30 for promoting the differentiation of stem cells into a neuronal phenotype, neuroblast proliferation, stem cell migration or stem cell axon outgrowth. A pharmaceutical composition comprising a N RP compound and a pharmaceutically acceptable medium ing the amino acid sequence of SEQ ID NO: 30.   An NRP compound consisting of the amino acid sequence of SEQ ID NO: 30.

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