Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• [0001] Provided are methods for promoting neuronal survival, axonal growth and/or regeneration and more specifically use of SKI-1 and/or furin inhibitors for promoting axonal growth and/or regeneration.
• Axonal outgrowth is precisely orchestrated during development to ensure correct connectivity within the nervous system (Tessier-Lavigne and Goodman, 1996). Surprisingly, however, there are a relatively limited number of known guidance proteins (Thanos and Mey, 2001 ). Post-translational modification is one strategy to generate multiple activities from a single protein. In theory, complex proteolytic processing with alternate use of multiple specific cleavage sites could dramatically increase diversity (Zisman et al., 2007).
• Semaphores which have several cleavage sites, are one example (Adams et al., 2007), but the extent to which this strategy is utilized and the effects on activity, receptor specificity, and/or short (membrane -bound) versus long-range (soluble) effects are largely unknown.
• the GPI- anchored protein RGMa is key to the development of various projections within the CNS and is thought to act solely as a membrane-bound protein (Monnier et al., 2002). Its activity is similar to that of the ephrins, as it inhibits retinal ganglion cell (RGC) outgrowth (Monnier et al., 2002). RGMa acts through the transmembrane receptor Neogenin, which is expressed in a high-temporal low-nasal gradient in RGC axons (Rajagopalan et al., 2004).
• Neogenin and RGMa provide positional information for retinal axons invading the tectum. Besides its contribution to the establishment of the neuronal architecture, RGMa is a major impediment to neuronal regeneration.
• the disclosure includes a method of inhibiting RGMa cleavage comprising contacting the RGMa with a proprotein convertase (PPC) inhibitor, optionally a Subtilisin Kexin lsoenzyme-1 (SKI-1) inhibitor and/or a furin inhibitor.
• PPC proprotein convertase
• SKI-1 Subtilisin Kexin lsoenzyme-1
• disclosure includes a method for inhibiting RGMa cleavage at
• RTFTJ.D amino acid 175 in human
• SKI-1 Subtilisin Kexin lsoenzyme-1
• inhibition of RGMa cleavage at RTFTjD inhibits formation of RGMa37.
• the SKI-1 inhibitor is a serine protease inhibitor, optionally a membrane permeable serine protease inhibitor.
• the RGMa is attached to a cell and the serine protease inhibitor is a membrane permeable serine protease inhibitor.
• the disclosure includes a method of inhibiting soluble NRGMa or
• NNRGMa release from a cell comprising contacting the cell with a PPC inhibitor such as a SKI-1 inhibitor and/or a furin inhibitor.
• a PPC inhibitor such as a SKI-1 inhibitor and/or a furin inhibitor.
• the inhibitor is a membrane permeable serine protease inhibitor.
• the PPC inhibitor is selected from a membrane permeable serine protease inhibitor, optionally AEBSF, an ER permeable serine protease inhibitor and RVKR peptide.
• the SKI-1 inhibitor is selected from RRLL peptide (SEQ ID NO: 1]
• Another aspect includes a method for stimulating neuron survival, axon growth and/or promoting neuron axon regeneration of a neural cell in a subject in need thereof, the method comprising, providing neural tissue with a PPC inhibitor, optionally a SKI-1 inhibitor and/or a furin inhibitor.
• the furin inhibitor is a furin prosegment inhibitor.
• the method is for inducing long range neuron survival, axon growth and/or regeneration.
• the neural cell is provided with the SKI-1 inhibitor and/or the furin inhibitor by administering the inhibitor to the subject.
• the subject has Multiple sclerosis.
• the subject has a nerve injury.
• the nerve injury is a spinal cord injury.
• the subject is suffering or has suffered an ischemic neuronal injury.
• the subject has glaucoma, Alzheimer's disease, Parkinson's disease and/or a CNS immune response.
• Also provided in another aspect is a method for stimulating neuron survival, axon growth and/or regeneration in a subject in need thereof, the method comprising administering to the subject a PPC inhibitor, optionally a SKI-1 and/or Furin inhibitor, in sufficient amount to promote neuron survival, axon growth and/or regeneration of the neuron.
• a further aspect includes treating nerve damage comprising administering to a subject in need thereof a composition comprising a PPC inhibitor such as a SKI-1 inhibitor and/or a Furin inhibitor.
• a PPC inhibitor such as a SKI-1 inhibitor and/or a Furin inhibitor.
• the nerve damage is a neurodegenerative disease.
• the neurodegenerative disease is multiple sclerosis or glaucoma.
• the nerve damage is a spinal cord injury, ischemic injury such as a stroke or CNS immune injury.
• the damaged nerve comprises a sensory neuron or a motor neuron.
• the method is for inhibiting an immune response in CNS tissues, e.g. brain and spinal cord in multiple sclerosis patients comprising administering a PPC inhibitor such as a SKI-1 inhibitor and/or a furin inhibitor.
• a PPC inhibitor such as a SKI-1 inhibitor and/or a furin inhibitor.
• the method is for inhibiting demyelination in a subject in need thereof comprising administering a SKI-1 inhibitor and/or a furin inhibitor.
• the SKI-1 inhibitor and/or the Furin inhibitor is administered to the lesion site directly. In another embodiment the inhibitor is administered systemically.
• the PPC inhibitor is selected from a membrane permeable serine protease inhibitor, optionally AEBSF, an ER permeable serine protease inhibitor and RVKR peptide.
• the SKI-1 inhibitor is selected from RRLL peptide (SEQ ID NO: 1]
• PF-429242 peptide CIYISRRLLC (SEQ ID NO: 5; optionally with terminal "C” residues cyclized) and/or prosegment inhibitor R134E.
• the inhibitor(s) and/or composition is/are administered by intravenous, intraspinal and/or intracranial infusion. In another embodiment he inhibitor(s) and/or composition is/are administered by Intraperitoneal and/or intrathecal application.
• a further aspect is an isolated polypeptide comprising: a RGMa fragment selected from or corresponding to:
• RGMa M68 a RGMa fragment no longer than amino acid 1 to 168 of human RGMa (RGMa M68 ), optionally no longer than amino acid 47 to 168,
• RGMa-i.127 a RGMa fragment no longer than amino acid 1 to 127 of human RGMa (RGMa-i.127), optionally no longer than amino acid 47 to 127,
• RGMa MT s a RGMa fragment no longer than amino acid 1 to 175 of human RGMa (RGMa MT s), optionally no longer than amino acid 47 to 175;
• the consists of: residues 1 to 168 of SEQ ID NO: 1 or residues 47 to 168 of SEQ ID NO: 1 ; consists of: residues 1 to 175 of SEQ ID NO: 1 or residues 47 to 175 of SEQ ID NO: 1 or fragments thereof, RGMai.i 33 consists of residues 1 to 133 of SEQ ID NO: 1 or residues 47 to 133 of SEQ ID NO: 1 ; RGMa 165 SO consists of residues 169 to 450 of SEQ ID NO: 1 ; RGMa 17 ⁇ 5o consists of residues 176 to 450 of SEQ ID NO: 1 ; RGMai 28 -450 consists of residues 128to 450 of SEQ ID NO: 1 ; or RGMa 2 oi-29o consists of residues 201 to 290 of SEQ ID NO: 1 , and/or a conservative variant thereof.
• the fragment is RGMa ⁇ 68 , RGMav ⁇ and/or RGMa 20 i-29o and/or a corresponding species fragment and/or a conservative variant thereof, wherein the conservative variant retains the ability to bind neogenin.
• the isolated polypeptide comprises RGMa128-175 in combination with a second fragment of RGMa, optionally no longer than amino acids 169 to 450 of human RGMa (RGMa169-450) wherein the isolated polypeptide is combined with the second fragment via a disulphide bridge forming a species that is approximately 37kDa when separated electrophoretically on a non-reducing agarose gel.
• the isolated polypeptide is glycosylated and/or further comprises a
• the isolated polypeptide comprises one or more of a linker peptide and a tag such as a FLAG ® -tag.
• an antibody that specifically binds an epitope comprising amino acid 175 of SEQ ID NO: 1 and/or blocks cleavage of RGMa at amino acid 75 by SKI-1 or minds a D168 or H170 mutant.
• the isolated peptide or the antibody is attached to a substrate.
• a further aspect includes a composition or kit comprising an isolated polypeptide, inhibitor or the antibody described herein, optionally for use in a method described herein.
• FIG. 1 Analysis of RGMa processing.
• A Schematic representation of the 7 RGMa peptides identified in the analysis. In membranes under reducing (+DTT) or non-reducing (-DTT) conditions, an anti C-RGMa revealed 4 RGMa fragments. In cell supernatant (-DTT), an anti-N- RGMa revealed that 3 RGMa fragments are released. Molecular weights are indicated on the right; UG unglycosylated. Black arrow head indicates autocatalytic cleavage site, black arrow represents known shedding cleavage site. Gray arrows indicate new calculated cleavage sites.
• FIG. 1 RGMa is processed differentially in SH-SY5Y and DF1 cells.
• RGMa transfected SH-SY5Y cell membranes displayed the same pattern as brain membrane with 3 fragments at 60kDa, 37kDa and 33 kDa under non reducing conditions and 2 bands at 33kDa and 60kDa under reducing conditions.
• FIG. 3 Furin and SKI-1 process RGMa and are involved in pathfinding.
• A RGMa expressing cells were treated with the protease inhibitors AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) or RVKR (SEQ ID NO: 3), or DMSO (control) and membranes blotted.
• AEBSF and RVKR both strongly reduced RGMa cleavages, shown by the reduction of the 33 and 37 kDa bands (Asterisks) (see also Figures 4A,B).
• B RGMa expressing cells were treated with RVKR or RRLL (SEQ ID NO:4) protease inhibitors or DMSO (control).
• RGMa was co-transfected with ppFurin or empty plasmid (Ctrl). ppFurin strongly reduced formation of the 37 kDa fragment, confirming that it results from Furin activity (see also Figure 4).
• E RGMa expressing cells were treated with DMSO (control) or RVKR and supernatants were blotted with anti-N-RGMa. RVKR suppressed release of N-terminal proteins (Asterisk).
• F-H In situ analysis demonstrated that Furin and SKI-1 are expressed in the tectum in RGMa expressing radial glial cells (arrows). Controls (sense) did not show any staining (H).
• FIG. 4 RGMa is processed by Proprotein Convertase Furin between the ER and the Golgi.
• RGMa was expressed in HEK cells in the presence of Brefeldin A and Golgicide A. In the presence of these inhibitors, RGMa was still processed by HEK cells (presence of the 33 and 37 kDa bands) indicating that RGMa processing occurs before it enters the Golgi.
• Binding is abolished in noncleavable mutants (D149A, H151A) versus wtRGMa (wt; see also Figures S3A-S3C).
• D Temporal explants grown on Mock, wtRGMa (wt), and noncleavable mutants (D149A; H151A) membranes. Axonal growth is reduced on wtRGMa versus Mock and mutants. Bar, 150 pm.
• mutation of the cleavage site abolished RGMa inhibition on temporal axons (**p ⁇ 0.05). Nasal axons that express less Neogenin were less inhibited.
• NIE-115 cells were grown on Mock, wtRGMa (wt), D149A, and H151A membranes, after transfection with GFP plasmid plus a control shRNA (Ctrl-shRNA) or a Neogenin shRNA (Neo-shRNA).
• Ctrl-shRNA control shRNA
• Neo-shRNA Neogenin shRNA
• NIE-115 cells transfected with Ctrl- shRNA extend shorter neurites on wtRGMa versus Mock, D149A, and H151A. Bar, 30 Mm.(G) D149A and H151A significantly increased length (**p ⁇ 0.001 ) compared to wtRGMa.
• Neogenin silencing with Neo-shRNA suppressed wtRGMa inhibition.
• FIG.A-C Cell surface localization of RGMa constructs in COS-7 cells.
• COS-7 cells were transfected with RGMa mutant proteins and RGMa localization was studied with an anti- C-RGMa antibody. Staining was done after paraformaldehyde fixation and without treatment with detergent to stain only cell surface proteins.
• A,B As indicated by the strong cell surface staining, the two cleavage mutant proteins H151A and D149A localize at the cell surface.
• C Control experiments in which Mock transfected cells were stained only show background staining.
• RGMa with a TEV site (B) Under non-reducing conditions, RGMa appears as a 60 kDa band, whereas RGMa-TEV is cleaved by TEV and only C-RGMa (33kDa) is apparent.
• C,D Neogenin-AP binds saturably to wtRGMa (C) and C-RGMa (D). Binding of Neogenin-AP or RGMa-AP to microtiter wells coated with C-RGMa and wtRGMa membranes. Neogenin-AP binding to BSA was less than 5% of these levels. Calculated Kd of Neogenin-AP is indicated. Data from 6 determinations.
• FIG. 8 The C-terminal part of RGMa, expression and function.
• C-RGMa 182"271 The presence of C-RGMa 182"271 strongly reduced axonal growth.
• C-F In vivo expression of C-RGMa 182"271 fragments was cloned as His-Tag proteins in RCAS viral vector to infect E1 .5 chick developing tecta. At E10, tecta were removed, fixed and sections stained with an anti-His antibody.
• B,D DAPI staining of the immunostaining presented in the right panels.
• C Sections from Mock (empty RCAS) infected embryos displayed background staining.
• E Sections from animals infected with RCAS constructs that expressed C-RGMa 182'271 showed a strong staining, which indicated that this fragment was expressed in the chick tecta.
• FIG. 9 Soluble RGMa proteins display Neogenin dependent inhibition.
• A RGMa proteins tested.
• B Temporal explants on laminin, laminin+5pg/ml N-RGMa, 2.5pg/ml NN-RGMa, and 10pg/ml RGMaA. The 3 RGMas inhibited growth. Bar, 200pm.
• C RGMa proteins significantly decreased growth (**p ⁇ 0.0001 ). Proteins displayed a concentration dependent effect, NN-RGMa having the strongest effect. Nasal axons were less inhibited. Data are average ⁇ SEM from 4 independent experiments.
• E RGMaA, N-RGMa, and NN-RGMa inhibited growth.
• Neo-shRNA increased the average neurite-length on N-RGMa, NN-RGMa, and RGMaA, when compared to Ctrl-shRNA (**p ⁇ 0.05). Data are average ⁇ SEM from 4 independent experiments.
• G,H,I Neogenin-AP (2 g/weW) bound saturably to purified RGMaA (G), N-RGMa (H), and NN-RGMa (I). RGMa-AP (2 pg/well) did not bind to RGMa proteins. Neogenin-AP binding to BSA was less than 5% of these levels. Kd of Neogenin-AP is indicated.
• Neogenin constructs were cloned with an His tag, expressed in COS-7 cells, purified on Nickel agarose, and separated in SDS-PAGE electrophoresis before staining with coomassie. All proteins used in our studies had an apparent purity of >90% after coomassie staining.
• RGMaA full length soluble RGMa
• B-C N-RGMa-AP
• B NN-RGMa- AP (C) binding to Neogenin.
• Microtiter wells were coated with purified extracellular domain of Neogenin at concentration from OnM to 80nM. Binding of N-RGMa-AP (C; 2 pg/well) or NN-RGMa- AP (D; 2 pg/well) to the coated wells were assessed after incubation at 20 °C for 3 h. Binding of both N-RGMa-AP and NN-RGMa-AP to BSA-coated wells was less than 5% of these levels. Calculated affinity of N-RGMa-AP and NN-RGMa-AP for the ectodomain of Neogenin is indicated on the graph.
• NN-RGMa displayed a mi:oh higher affinity to Neogenin when compared to N-RGMa. Data are the mean ⁇ SEM from 6 determinations.
• Figure 11 Over-expression of soluble RGMa proteins perturbs axonal pathfinding in the embryonic chicken visual system. At embryonic day 1.5 (E1.5), RCAS-virus (control) and RCAS-RGMa (positive control), RCAS-N-RGMa, RCAS-NN-RGMa were injected into the developing optic tectum. At E15, a Dil crystal was placed in the temporal retina to label fibers. (A) In control experiments, all axons converge towards a well-defined terminal zone (TZ).
• TZ well-defined terminal zone
• the insets represent a drawing of the flat- mounted retina to indicate the location of the Dil crystal in a dorso-temporal position of the retina and the path of axons towards the optic fissure, t, temporal; d, dorsal; v, ventral; n, nasal.
• the hypothetical TZ is represented in each panel (see also Figure 12).
• FIG. 12 In vivo expression of N-RGMa and NN-RGMa: N-RGMa and NN-RGMa fragments were cloned as His-Tag proteins in RCAS viral vector to infect E1.5 chick developing tecta. At E10, tecta were removed, fixed and sections stained with an anti-His antibody. A,C,E) DAPI staining of the immunostaining presented in the right panels. (B) Sections from Mock (empty RCAS) infected embryos displayed background staining.
• N- and C-RGMa inhibit axonal outgrowth by binding to the same Neogenin domain.
• the extracellular portion of Neogenin consists of 4 Immunoglobulin like (4lg) and 6 fibronectin type III (6FNIII) domains.
• ELISA plates were coated with RGMa proteins and binding of Neogenin-AP was studied.
• - base line; +> 2 x base line; ++ > 3 x base line; +++> 5x base line).
• the 3-4 fibronectin (FNIII(3-4)) domains sufficed for binding to RGMa proteins.
• Figure 14 Western blot showing RGMa release after treatment with RRLL or RVKR.
• FIG. 15 SKI-1 inhibitor reduces cerebral infarct volume and edema and improves functional outcome following stroke.
• A Representative brain slices showing infarct area in Control and SKI-1 inhibitor groups after focal cerebral ischemia in rat. Quantification of (B) brain infarct volume and (C) edema in Control and SKI-1 inhibitor-treated animals.
• D Quantification of neurological outcome using Bederson's test at different time points in Control and SKI-1 inhibitor treated groups after focal cerebral ischemia in rat. Data are means + SEM; * p ⁇ 0.05 vs. Control.
• Temporal retinal explants cultured on supernatant from cells transfected with Mock (Control; called Laminin in figure) or RGMa and treated with either DMSO or 10 ⁇ PF429242 (SKI-1 inhibitor). After overnight incubation, membranes from Control, RGMa+DMSO and RGMa+PF429242 cells were prepared and used as a substrate for growing retinal fibers. Retinal explants were put on membranes with laminin and cultured overnight before fixation, then stained with Alexa-488 phalloidin and number of fibers/explant was measured and neurite length was quantified. Quantification of (A) number of fibers and (B) neurite length. Data are average ⁇ SEM. * p ⁇ 0.05 vs.
• a cell as used herein includes a single cell as well as a plurality or population of cells.
• a neuron as used herein includes a single cell as well as a plurality or population of neurons.
• administered means administration of a therapeutically effective dose of a compound or composition of the disclosure to a cell either in cell culture or in a subject e.g. patient.
• antibody as used herein is intended to include monoclonal antibodies, polyclonal antibodies, and chimeric antibodies.
• the antibody may be from recombinant sources and/or produced in transgenic animals.
• Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
• Antibody fragments mean binding fragments.
• Antibodies capable of binding may be prepared by conventional methods.
• a mammal e.g. a mouse, hamster, or rabbit
• Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art.
• the peptide can be administered in the presence of adjuvant.
• the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
• Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies.
• antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
• antibody producing cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
• myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
• somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
• Such techniques are well known in the art, (e.g. the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol.
• Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.
• the term "conservative variant” refers to a variant of a polypeptide such as a variant of a RGMa fragment, which comprises one or more amino acid substitutions or deletions (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more, optionally 11 -20, 21-30 or more, for example upto 0% of a polypeptide or nucleic acid) that do not substantially affect the character of the variant polypeptide relative to the starting polypeptide.
• polypeptide character is not substantially affected if the substitutions or deletions do not preclude specific binding of the variant peptide to a specific binding partner of the starting polypeptide.
• conservative amino acid substitutions for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more, optionally 11 -20, 21-30 or more, for example upto 0% of a polypeptide or nucleic acid
• Conservative mutations may be made for example in non-Neogenin binding regions and in non cleavage domains, and include for example conservative changes that do not impact RGMa axon inhibition activity or RGMa survival inducing activity determinable for example using an assay described herein.
• active fragment means a portion of a RGMA fragment such as RGMa 16 9. 450 that is immunogenic, binds neogenin, promotes neuron cell survival and/or inhibits axon outgrowth
• furin as used herein means a member of the subtilisin-like proprotein convertase family, having typically RX(K/R)R consensus motif and includes without limitation all known furin molecules including naturally occurring variants and for example those deposited in Genbank with the accession numbers CAA27860 CAA27860.1 , CAA37988.1 , NP_062204.1 , NP_003782.1 , NP_001 161382.1 , which are specifically incorporated by reference. Furin is also known as Pace and PC1 , PCSK3 SPC1.
• furin inhibitor refers to any peptide, small molecule or other inhibitor that reduces furin enzymatic activity by at least 40%, 50%, 60%, 70%, 80%, 90% or more.
• Non-limiting examples include furin inhibitors disclosed in U.S. Patent Application Publication No. 20090131328 titled “FURIN INHIBITORS and/or U.S. Patent Application Publication No. 20110059896 titled "USE OF FURIN CONVERTASE INHIBITORS IN THE TREATMENT OF FIBROSIS AND SCARRING" each of which are specifically incorporated by reference.
• Non-limiting examples of furin inhibitors include peptidyl chloroalkylketones with peptide moieties that mimic the convertase enzyme cleavage site, such as decanoyl-RVKR-cmk (also referred to as RVKR (SEQ ID NO:3) and derivatives thereof.
• the inhibitors include modifications that improve membrane solubility such as attaching a moiety such as for example a decanoyl moiety or an HIV TAT peptide, as is known in the art.
• Serine proteases and/or other PPC inhibitors that reduce furin enzymatic activity by at least 40%, 50%, 60%, 70%, 80%, 90% or more are also contemplated.
• the furin inhibitor can also for example be a furin-prosegment inhibitor.
• furin- prosegment inhibitor refers to an inactive form or fragment of furin that comprises an N-terminal prosegment that acts as a dominant negative form of Furin.
• Proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that has been suggested to act both as an intramolecular chaperone and an inhibitor of its parent enzyme.
• Furin prosegment is a potent inhibitor of furin as are small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors.
• the fragment can be for example at least 10-20 amino acids, for example 0 amino acids and/or including any one of the furin prosegment derived peptides described in Table 2 of Zhong et al 1999 and/or US Patent 7,211 ,424 (Seidah et al), each herein specifically incorporated by reference.
• isolated polypeptide refers to a proteinaceous agent, such as a peptide, polypeptide or protein, which is substantially free of cellular material or culture medium when produced recombinantly, or chemical precursors, or other chemicals, when chemically synthesized.
• polypeptide refers to a sequence of amino acids consisiting of naturally occurring residues, and non-naturally occurring residues.
• isolated nucleic acid refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
• An "isolated nucleic acid” is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.
• nucleic acid as used herein includes a sequence of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages, and is intended to include DNA and RNA and can be either double stranded or single stranded represent the sense or antisense strand.
• proprotein convertase inhibitor and or "PPC” as used herein refers to any peptide, polypeptide, small molecule or other inhibitor that reduces furin and/or SKI-1 enzymatic activity by at least 40%, 50%, 60%, 70%, 80%, 90% or more, and includes for example serine protease inhibitors, optionally a membrane permeable serine protease inhibitor, such as AEBSF (4- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), RVKR peptide (SEQ ID NO:3), decanoyl- RVKR-cmk and derivatives, specific SKI-1 inhibitors and/or furin inhibitors as well as inhibitors that inhibit both SKI-1 and furin and optionally other PPCs.
• AEBSF 2- (2-Aminoethyl) benzenesulfonyl fluoride hydrochloride
• RVKR peptide SEQ ID NO:3
• the inhibitors include modifications that improve membrane solubility such as attaching a moiety such as a decanoyi moiety or a HIV TAT peptide.
• Polypeptide PPCs include for example prosegment inhibitor R134E which inhibits SKI-1 , as well as furin- prosegment inhibitor which inhibits furin.
• prosegment inhibitor R134E refers to an inactive form of
• R134E SKI-1 that acts as a dominant negative inhibitor.
• R134E is in a conserved motif across species including human and murine.
• the term "providing neural tissue comprising neurons and RGMa expressing cells with a PPC inhibitor, optionally a SKI-1 inhibitor and/or a furin inhibitor” means providing the PPC inhibitor or optionally a SKI-1 inhibitor and/or a furin inhibitor, to a site where RMGa expressing cells are present and in contact with neurons (e.g. signaling contact and/or physical contact) and where inhibition of RGMa cleavage in the RGMa expressing cell interrupts RGMa axonal outgrowth inhibition, thereby allowing neuronal axonal growth and/or regeneration.
• RGMa is expressed by astroglia cells and oligodendrocytes, which are the cells that secrete inhibitors that hamper axonal regeneration (Schwab et al., 2005).
• the PPC inhibitor can be provided to the neural tissue by a number of methods depending on the location of the neural tissue.
• the PPC inhibitor optionally the SKI-1 inhibitor can be provided by injection, for example to a site of injury, such as a spinal cord lesion or a stroke infarct lesion.
• the inhibitor could be administered for example, intrathecally (e.g. using osmotic pumps) or by systemic application.
• the cells are contacted with the PPC inhibitor in vitro.
• the neural tissue is neogenin expressing neural tissue.
• RGMa refers to Repulsive Guidance Molecule A and includes without limitation all known RGMa molecules including naturally occurring variants and for example those deposited in Genbank with accession numbers AAH15886.2, CAD89718.1 , NP- 001100994.1 , and/or NP-989868.1 , which are herein specifically incorporated by reference as well as conservative amino acid containing forms and functional mutants (e.g. mutations that have a functional effect), for example those described herein.
• a human amino acid sequence of RGMa is provided in SEQ ID N0.1.
• the sequence provided by SEQ ID NO:1 includes a signal peptide comprising amino acids (aa) 1 to 46.
• RGMa is cleaved for example providing a N-terminal RMGMa which is aa 47-168, C-terminal is aa. 169-450 including the GPI anchor sequence as well as other fragments described herein.
• Mutant forms include for example, RGMa mutated at residues within the SKI-1 and furin cleavage sites (e.g. residue 175 of SEQ ID N0.1 ) (e.g. uncleavable mutants) and forms comprising mutated residues flanking the autocatalytic site (e.g. H151A or D149A in chick, corresponding human positions being D168A and H170A).
• RGMa fragment refers to a RGMa fragment selected from or corresponding to:
• RGMa fragment no longer than amino acid 1 to 168 of human RGMa (RGMai.i68). optionally no longer than amino acid 47 to 168,
• RGMa 1-127 a RGMa fragment no longer than amino acid 1 to 127 of human RGMa (RGMa 1-127 ), optionally no longer than amino acid 47 to 127,
• n a RGMa fragment no longer than amino acid 1 to 133 of human RGMa (RGMai. 133 ), optionally no longer than amino acid 47 to 133,
• RGMa 128 .45o a RGMa fragment no longer than amino acid 128 to 175 of human RGMa q) a RGMa fragment no longer than amino acid 169 to 450 of human RGMa r) a RGMa fragment no longer than amino acid 175 to 450 of human RGMa s) a RGMa fragment no longer than amino acid 128 to 450 of human RGMa (RGMa 128 .45o)
• the amino acid numbers can be e.g. 1 to 168 for in reference to the human amino acid sequence (e.g. SEQ ID NO:1 ).
• a person skilled in the art would readily be able through sequence alignment, be able to determine the corresponding species fragments e.g. determine the amino acid positions of corresponding fragments in other species.
• the corresponding amino acids for the RGMa fragment no longer than amino acids 1 to 168 of human RGMa corresponds to amino acids 1 to 150 in chick RGMa; and for the RGMa fragment no longer than amino acids 201-290 of human RGMa corresponds to amino acids 182-271 in chick RGMa.
• the fragment can be any length within the stipulated range or the full length of the stipulated range.
• RGMa ⁇ ee fragment can be about 80, 90, 100, 110, 120, 122, 130, 140, 150 160, or 168 or any number of amino acids in between 10 and 168 wherein the fragment retains neogenin binding and/or signaling activity (for example including or excluding the signal sequence).
• Full length RGMa!. 16 8 corresponds for example to the 22 kDa fragment illustrated in Figure 1.
• RGMa!.,27 fragment can be about 60, 70, 80, 87, 90, 100, 110, 120, or 127 amino acids or any number of amino acids in between 10 and 127 wherein the fragment is antigenic, retains neogenin binding and/or signaling activity (and can for example including or excluding the signal sequence).
• RGMa-M3 3 fragment which was initially identified and led to determination of the furin cleavage site, can be about 60, 70, 80, 87, 90, 100, 110, 120, 130 or 133 amino acids or any number of amino acids in between 10 and 133 wherein the fragment retains neogenin binding and/or signaling activity for example including or excluding the signal sequence).
• RGMa 1-175 fragment can be about 80, 90, 100, 110, 120, 129, 130, 140, 150 160, 170 or 175 or any number of amino acids in between 10 and 175, or any number of amino acids in between 10 and 175 wherein the fragment is antigenic, retains neogenin binding and/or signaling activity.
• RGMa 128 - 175 fragments can be about 10, 20, 30, 40 or 46 amino acids or any number of amino acids in between 10 and 45 wherein the fragment is antigenic.
• RGMa 76 _ 45 o fragments can be about 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 274 amino acids or any number of amino acids in between 10 and 274 wherein the fragment retains neogenin binding and/or signaling activity.
• RGMa 169 -45o fragments can be about 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 281 amino acids or any number of amino acids in between 10 and 281 wherein the fragment retains neogenin binding and/or signaling activity.
• Full length RGMa 169 .
• RGMa 12 s-45o fragments can be about 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320 or 322 amino acids or any number of amino acids in between 10 and 322 wherein the fragment retains neogenin binding and/or signaling activity.
• RGMa 176 _ 2 9o fragments can be about 40, 50, 60, 70, 80, 90, 100, 110 or 114 amino acids or any number of amino acids in between 10 and 114 wherein the fragment retains neogenin binding and/or signaling activity
• RGMa 20 i-29o fragments can be about 40, 50, 60, 70, 80 or 89 amino acids or any number of amino acids in between 10 and 89 wherein the fragment retains neogenin binding and/or signaling activity.
• RGMaA or "RGMa delta” as used herein refers to a 60 kDa species that results from RGMa shedding from the membrane (RGMaA) by a phospholipase.
• RGMa is a membrane protein, however, soluble full length protein is found in RGMa transfected cell supernatant, indicating that it can be released.
• RGMa37 refers to a RGMa polypeptide, optionally membrane bound, which is schematically illustrated in Figure 1. It is a 37 kDa species (“RGMa37”) which is observed on non-reduced membrane preparations from chick brain as described in Example 1 , and can include for example a Cterm RGMa 33kDa fragment in combination with a 4kDa RGMa fragment, which are associated via a disulphide bridge. RGMa37 corresponds to amino acids about 128-450 or optionally aboutl 34-450 of human RGMa (for example plus or minus 8 amino acids and starting optionally at 126).
• RGMa ⁇ ee (e.g.22 kDa fragment) also referred to as "N-RGMa”, as used herein means a RGMa fragment no longer than amino acid 1 to 168 of human RGMa, for example about 80, 90, 100, 110, 120, 122, 130, 140, 150 160, or 168 or any number in between 10 and 168 that retains neogenin binding and/or signaling activity.
• Full length RGMa M6 8 (and/or RGMa deleted of the signal sequence e.g. corresponding to aa 47-168) corresponds to the 22 kDa fragment illustrated in Figure .
• RGMa ⁇ s e.g.18kDa fragment
• N-RGMa a RGMa fragment no longer than amino acids 1 to 133 of human RGMa, for example about 60, 70, 80, 87, 90, 100, 1 10, 120, 130 or 133 amino acids or any number of amino acids in between 10 and 133 wherein the fragment retains neogenin binding and/or signaling activity.
• Full length RGMa,. ⁇ (and/or RGMa deleted of the signal sequence e.g. corresponding to aa 47-133) corresponds to the 18 kDa fragment illustrated in Figure 1.
• "RGMa-).- ⁇ " was used to identify the furin cleavage site that results in "RGMa ! . ⁇ ".
• N-RGMa means a RGMa fragment no longer than amino acids 1 to 127 of human RGMa, for example about 60, 70, 80, 86, 90, 100, 110, 120, or 127 amino acids or any number of amino acids in between 10 and 127 wherein the fragment is antigenic and/or retains neogenin binding and/or signaling activity.
• Full length RGMai. 2 7 is amino acids 1 -127 of SEQ I D NO:1 and/or deleted of the signal sequence e.g. corresponding to aa 47-127.
• Full length RGMa ⁇ s (and/or RGMa deleted of the signal sequence e.g. corresponding to aa 47-127) corresponds to the 18 kDa fragment illustrated in Figure 1.
• RGMa ⁇ s means a RGMa fragment no longer than amino acid 1 to 175 of human RGMa, for example about 80, 90, 100, 110, 120, 129, 130, 140, 150 160, 170 or 175 or any number in between 10 and 175 that retains neogenin binding and/or signaling activity.
• RGMai_ 17 5 and/or RGMa deleted of the signal sequence e.g. corresponding to aa 47-175 is considered full length RGMai-i 75 .
• GMa ! 28-175 '. means a RGMa fragment no longer than amino acid 128 to 175 of human RGMa, for example about 10, 20, 30, or 47 or any number in between 10 and 47 that is antigenic. This fragment corresponds to for example the 4kDa RGMa fragment in Figure 1.
• RGMa ! 69-45o means a RGMa fragment no longer than amino acids 169 to 450 of human RGMa, for example about 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 281 amino acids or any number of amino acids in between 10 and 281 wherein the fragment retains neogenin binding and/or signaling activity.
• Full length RGMai 69- 50 corresponds to the C-terminal molecule of RGMa illustrated in Figure 1.
• RGMa ! 76-45o means a RGMa fragment no longer than amino acids 176 to 450 of human RGMa, for example about 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270 or 274 amino acids or any number of amino acids in between 10 and 274 wherein the fragment retains neogenin binding and/or signaling activity.
• RGMa 12 8- 4 5o means a RGMa fragment no longer than amino acids 128 to 450 of human RGMa, for example about 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320 or 322 amino acids or any number of amino acids in between 10 and 322 wherein the fragment retains neogenin binding and/or signaling activity.
• Full length RGMa 12 8-45o corresponds to a C-terminal molecule of RGMa illustrated in Figure 1.
• RGMa 176 -29o is in reference to human RGMa and means a
• RGMa fragment a RGMa fragment no longer than 176 to 290 of human RGMa, for example the human RGMa sequence of SEQ ID NO:1.
• RGMa 176 . 290 can be about 40, 50, 60, 70, 80, 90, 100, 110 or 114 amino acids or any number of amino acids in between 10 and 114 wherein the fragment retains neogenin binding and/or signaling activity.
• RGMa 2 oi-29o is in reference to human RGMa and means a
• RGMa 20 i-29o can be about 40, 50, 60, 70, 80 or 89 amino acids or any number of amino acids in between 10 and 89 wherein the fragment retains neogenin binding and/or signaling activity.
• sequence identity refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
• the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
• a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877.
• Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402.
• PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
• the default parameters of the respective programs e.g., of XBLAST and NBLAST
• the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
• SKI-1 as used herein means Subtilisin Kexin lsozyme-1 , a member of the subtilisin-like proprotein convertase family, and includes without limitation all known SKI-1 molecules including naturally occurring variants and for example those deposited in Genbank with accession number AAD27010.1 , AAD27011.1 , AAI14556.1 , ABT02354.1 , XP_003641945, which are herein specifically incorporated by reference. SKI-1 is also known for example as site 1 Protease, and S1 P. It is not possible to predict the presence of a SKI-1 cleavage sites in RGMa sequence, for example using prediction programs, with any certainty.
• SKI-1 The concensus sequence recognized by SKI-1 is reported as (R/K)X(hydrophobic)Zl where Z is variable (Seidah and Chretien, 1999). SKI-1 cleaves RGMa at amino acid 175 of SEQ ID NO:1.
• SKI-1 inhibitor refers to any peptide, polypeptide, small molecule or other inhibitor that reduces SKI-1 enzymatic activity by at least 40%, 50%, 60%, 70%, 80%, 90% or more.
• Non-limiting examples include, RRLL peptide (SEQ ID NO:4), PF-429242 (Pasquato et al, 2012) available for example from TOCRIS Science, peptide CIYISRRLLC (SEQ ID NO:5; optionally with terminal "C” residues cyclized) (Pasquato et al, 2011 ) and/or prosegment inhibitor R134E.
• the SKI-1 inhibitors include modifications that improve membrane solubility such as attaching a moiety such as a decanoyi moiety or a HIV TAT peptide.
• Serine proteases and/or other PPC inhibitors that reduce SKI-1 enzymatic activity by at least 40%, 50%, 60%, 70%, 80%, 90% or more are also contemplated.
• soluble RGMa as used herein means non-membrane bound forms of
• RGMa including for example N-RGMa, NN-RGMa and RGMaA, as well as the 4kDa RGMa fragment and/or fragments of any thereof.
• treatment is an approach for obtaining beneficial or desired results, including clinical results.
• beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
• Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
• a "therapeutically effective amount”, “effective amount” or a "sufficient amount” of an inhibitor(s) (e.g. SKI-1 and/or Furin inhibitor) or a composition comprising the inhibitor is a quantity sufficient to, when administered to a cell or a subject, including a mammal, for example a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount” or synonym thereto depends upon the context in which it is being applied.
• RGMa Fibronectin III
• an aspect of the disclosure includes an isolated polypeptide comprising: a RGMa fragment selected from or corresponding to:
• the RGMa ⁇ ee consists of residues 1 to 168 of SEQ ID NO:1 or a fragment thereof, for example deleted of the signal sequence;
• RGMai. 133 consists of residues 1 to 133 of SEQ ID NO:1 or a fragment thereof for example deleted of the signal sequence,
• RGMai_ 1 2 7 consists of residues 1 to 127 of SEQ ID NO:1 or a fragment thereof for example deleted of the signal sequence,
• RGMa 1 -17 5 consists of residues 1 to 175 of SEQ ID NO:1 or a fragment thereof for example deleted of the signal sequence;
• RGMa 128 . 175 consists of residues 128 to 175 of SEQ ID NO:1 or a fragment thereof, RGMa 169 .
• RGMa 128 -4 5 o consists of residues 128 to 450 of SEQ ID NO;1 or a fragment thereof, RGMa 176 .
• 450 consists of residues 176 to 450 of SEQ ID NO:1 or a fragment thereof;
• RGMai 76-290 comprises residues 176 to 290 of SEQ ID NO:1 or a fragment and/or RGMa 2 oi- 2 9o comprises residues 201 to 290 of SEQ ID N0.1 or a fragment thereof; and/or a conservative variant of any thereof.
• the RGMa fragment is another species corresponding fragment, e.g.
• Fragments that bind Neogenin include for example, N-RGMa (1-168 for human and 1-150 for chick), NN-RGMa (1-133 or 1-128 in human and 1 -1 14 in chick), (as well as fragments that are deleted of the signal peptide) and C terminal -RGMa fragments comprising of 201 to 290 in humans is 182-271 in chick.
• RGMa fragments 1-175 (and deleted of the signal peptide) and 176- 450 are also expected to bind Neogenin.
• the fragment is full length RGMai. 168 , RGMa 1 . 175 , RCMa ⁇ , and/or RGMai. 2 e, and/or a conservative variant thereof, and/or the fragment deleted of its signal peptide (e.g. residues 1 -46 are deleted from RGMa 1 - 6 8, RGMa v 28, RGMai_ 175 or RGMa ⁇ s) wherein the conservative variant retains for example the ability to bind neogenin.
• the fragment deleted of its signal peptide e.g. residues 1 -46 are deleted from RGMa 1 - 6 8, RGMa v 28, RGMai_ 175 or RGMa ⁇ s
• the fragment is full length RGMa 20 i-29o
• the fragment is RGMai 2 8-i75
• the isolated polypeptide comprises a RGMa fragment such as the 4 kDa RGMa fragment in combination with a second fragment of RGMa, optionally the 33 kDa fragment having the combined sequence of RGMa37; wherein the isolated polypeptide is optionally combined with the second fragment via a disulphide bridge forming a species that is approximately 37kDa when separated electrophoretically on a non-reducing agarose gel.
• the combination comprises RGMa 16 9-45o for example of Figure 1.
• the isolated polypeptide comprises RGMa 169 . 45 o (RGMa Cterm fragment 169-450 in humans) in combination with a second fragment of RGMa, optionally no longer than amino acids 1 to 168 of RGMa (RGMa1 -168 in humans, optionally minus the signal sequence), wherein the isolated polypeptide is optionally combined with the second fragment via a disulphide bridge forming a species that is approximately 60kDa when separated electrophoretically on a non-reducing agarose gel.
• the isolated polypeptide comprises RGMa 176 . 450 (RGMa Cterm fragment 176-450 in humans) in combination with a second fragment of RGMa, optionally no longer than amino acids 1 to 175 of RGMa (RGMa1 -175 in humans, optionally minus the signal sequence), wherein the isolated polypeptide is optionally combined with the second fragment, for example a fragment described herein.
• the RGMa fragments are demonstrated to be glycosylated and as shown in Figure 1 to comprise GPI anchors (e.g. Cterminal fragments). Accordingly, in an embodiment, the isolated polypeptide is glycosylated. In another embodiment, the isolated polypeptide comprises a GPI anchor or a part thereof.
• the RGMa fragment can for example comprise a tag such as a His tag, a FLAG -tag or a fluorescent tag (e.g. GFP) or an enzyme tag such as alkaline phosphatase (AP).
• the tag can for example be linked to the RGMa fragment via a linker peptide, for example, a linker peptide comprising 6, 7, or 8 amino acids. Linker peptides are commonly employed so when attaching a tag so that the native polypeptide folding is maintained.
• isolated polypeptides comprising cleavage site RGMa mutants selected from D168A, H170A, (SEQ ID NO: 8) and mutation at 175.
• a cleavage site can also be replaced.
• RGMA is provided wherein RGMa CGLFGDP residues (residues 8-14 of SEQ ID NO: 7 are replaced with ENLYFQS (residues 8-14 of SEQ ID NO:6) (e.g. using AA-PNYTHCGLFGDPHLRTFTD (SEQ ID NO:7) and AA- PNYTHENLYFQSHLRTFTD (SEQ ID NO:6).
• the replaced sequence is for example provided in SEQ ID NO: 9.
• the isolated polypeptide comprises one or more of a linker peptide and a tag such as a FLAG ® -tag, GST tag, His Tag, GFP tag.
• the isolated polypeptides can be provided as recombinant polypeptides and used for example as standards in a kit and/or as ligands for studying neogenin signal transduction.
• Isolated polypeptides can be made by transfecting a cell such as a bacterial cell or an insect cell with a nucleic acid molecule encoding the polypeptide.
• nucleic acid molecules to a cell or cell population, including, for example, calcium phosphate transfection, DEAE-dextran transfection, infection, electroporation, lipofection, heat shock, magnetofection, nucleofection, integrating episome, use of a gene gun or microinjection.
• Introduction of nucleic acid molecules to a cell or cell population refers to both stable and transient uptake of the genetic material.
• a further aspect includes an isolated nucleic acid molecule encoding an isolated polypeptide described herein.
• an antibody was generated using a rat RGMa peptide corresponding to residues 309-322 (which correspond to human RGMa residues 308-321). Accordingly in an embodiment, the antibody is an antibody that is capable of binding amino acids 309- 322 of rat RGMa and/or amino acids 308-321 of human RGMa.
• SKI-1 is responsible for cleaving RGMa at amino acid residue 175 of SEQ ID NO: 1 , and/or amino acids corresponding thereto.
• a further aspect provides an antibody that specifically recognizes an epitope that comprises amino acid residue 175 of SEQ ID NO: 1 or a corresponding amino acid.
• the isolated antibody is a monoclonal antibody and/or a humanized antibody.
• the epitope can comprise for example 4 or more amino acids surrounding residue 175, and the antibody can be generated using a peptide comprising 4 or more amino acids centered (or comprising) residue 175 of SEQ ID NO:1. Larger epitopes can be used, for example comprising any where from 5 to 25 residues or more, where antibodies that are capable of biding an epitope comprising residue 175 of SEQ ID NO:1 are selected from example using affinity chromatography.
• Also provided is a method of producing an antibody comprising administering a RGMa antigenic peptide comprising for example at least 6 contiguous amino acids of a RGMa fragment described herein, optionally comprising for example a mutation site described herein, to a non-human mammal and isolating antibodies capable of binding the administered antigenic peptide.
• the isolated polypeptide and/or antibody can for example be attached to a substrate, such as a plate well or bead.
• a further aspect includes a composition comprising the isolated antibody and/or isolated polypeptide and/or the isolated nucleic acid.
• the composition can comprise for example a suitable vehicle or diluent.
• the suitable vehicle or diluent is a pharmaceutically suitable vehicle or diluent.
• proprotein convertases Furin and SKI-1 combine with autocatalytic-cleavage and a disulphide-bridge to generate four membrane-bound and three soluble forms of the Repulsive Guidance Molecule (RGMa).
• RGMa Repulsive Guidance Molecule
• a further aspect includes a method of inhibiting RGMa cleavage comprising contacting the RGMa with a proprotein convertase (PPC) inhibitor.
• PPC proprotein convertase
• the PPC inhibitor comprises a Subtilisin Kexin lsoenzyme-1 (SKI- 1 ) inhibitor and/or a furin inhibitor.
• the RGMa is GPI anchored to a cell.
• An embodiment includes a method of inhibiting RGMa cleavage at RTFT
• a further embodiment includes a method of inhibiting RGMa cleavage at RLR, comprising contacting the RGMa with a furin inhibitor.
• RGM protiens including RGMa, RGMb, and RGMc.
• RGMc also known as hemojuvelin, (having for example, Accession: NP_998818.1 Gl: 47458048 and/or Q6ZVN8.1 ) is also cleaved by furin. Accordingly methods described herein comprising inhibiting furin can be used for example with RGMc as well.
• RGMc is involved in hemochromatosis which is a blood iron condition.
• the methods and compositions described herein can be used to inhibit RGMc cleavage, for example in a subject afflicted by hemochromatosis.
• Inhibition of SKI-1 and/or furin mediated cleavage of RMGa inhibits formation of RGMa37 and/or release of, RGMa ⁇ e (e.g. 22kDa fragment) and/or RGMai. 133 RGMa-
• another aspect includes a method of inhibiting soluble NRGMa (RGMa ⁇ 168 (e.g. 22kDa fragment)) or NNRGMa (e.g. RGMa1 -128 18kDa fragment) release from a cell (e.g. a RGMa expressing cell) comprising contacting the cell with a PPC inhibitor, optionally a SKI-1 inhibitor and/or a furin inhibitor.
• a PPC inhibitor optionally a SKI-1 inhibitor and/or a furin inhibitor.
• contacting can be performed for example by administering the PPC inhibitor to the SL&ject, for example intravenously and/or to the site of RGMa expressing cells.
• Nterm RGMa fragments and Cterm RGMa fragments bind neogenin fibronectin domain, specifically fibronectin domain 3-4.
• RGMa fragments, neogenin and/or fibronectin 3-4 domain comprising fragments can be used to screen for compounds that decrease or increase RGMa and neogenin interaction and/or that promote neuron survival, axon growth and/or regeneration.
• an assay for identitying compounds that inhibit RGMa:neogenin binding and/or activation comprising:
• test compound interferes with RGMa Neogenin binding and/or activation.
• the assay can for example be performed in cell frees assay using polypeptides described herein and/or using cells expressing the polypeptides described herein.
• the test comound may be added prior to the addition of the RGMa fragment.
• Determining whether the test compound interferes with RGMa:Neogenin binding can comprise for example determining if the RGMa fragment polypeptide binds to the neogenin comprising fibronectin domain 3-4 polypeptide with the same affinity or the same extent. This can be assessed using a number of techniques known in the art for assessing protein protein interaction such as immunoprecipitation, yeast 2-hybrid systems and GST protein interaction.
• Determining whether the test compound interferes with RGMa Neogenin activity can be performed for example using cells expressing a Neogenin receptor, contacting the cells with soluble RGMa (or tethered RGMa), and the test comound and assessing for example axon growth in the presence and absence of test compound using for example methods as described herein.
• retinal explants can be tested in the presence of the test compound plus from cells that were either Mock transfected or transfected with full length RGMa.
• Additional controls can be used for example RGMa fragments and/or neogenin fragments and/or mutants that are not involved in RGMa: neogenin binding.
• Inhibiting RGMa cleavage and for example inhibiting release of soluble RGMa can be used to stimulate neuron survival, axon growth and/or regeneration and/or inhibit ischemic injury.
• FIG. 16 demonstrates SKI-1 inhibition restores the deleterious effect of RGMa on fibre number and axonal outgrowth.
• a further aspect includes a method of stimulating neuron survival, axon growth and/or promoting neuron axon regeneration and/or inhibiting ischemic injury (e.g. of neural tissues) in a subject in need thereof, the method comprising: providing neural tissue with a PPC inhibitor, optionally a SKI-1 inhibitor and/or a furin inhibitor.
• the neuron is a neogenin expressing neuron.
• the neural tissue comprises for example neurons and RGMa expressing cells.
• Providing the neural tissue with the PPC inhibitor for example by administration to the subject, optionally by injection (for example intravenously or by injection into the neural tissue e.g. intraspinal injection), to promote axon survival, growth and/or axon regeneration, for example post-natal axon survival, growth and/or regeneration, by inhibiting RGMa anti-outgrowth signals.
• a PPC inhibitor optionally a SKI-1 inhibitor and/or a furin inhibitor for stimulating neuron survival, axon growth and/or promoting neuron axon regeneration and/or inhibiting ischemic injury.
• a further embodiment comprises a PPC inhibitor, optionally a SKI-1 inhibitor and/or a furin inhibitor for use in stimulating neuron survival, axon growth and/or promoting neuron axon regeneration and/or inhibiting ischemic injury.
• the PPC inhibitor can for example be comprises in a composition, for example comprising a pharmaceutically acceptable diluent or carrier.
• Neogenin expressing neurons include for example cortico-spinal neurons, dorsal root ganglion (DRG) neurons, Retinal Ganglion Cells, and hippocampal neurons.
• RGMa expressing cells include for example retinal ganglion cells, DRGs, hippocampal, cortico-spinal neurons. Tissues comprising said cells, and/or diseases and/or injuries involving said cells can be treated with a a PPC inhibitor such as a SKI-1 and/or inhibitor to promote neuron survival, axon growth and/or regeneration and/or treat the disease involving said cells.
• a PPC inhibitor such as a SKI-1 and/or inhibitor to promote neuron survival, axon growth and/or regeneration and/or treat the disease involving said cells.
• Another aspect includes a method of promoting neuron survival, axon growth and/or regeneration in a subject in need thereof, the method comprising the step of contacting neural tissue with a PPC inhibitor, optionally a SKI-1 and/or Furin inhibitor in an amount sufficient to promote neuron survival, axon growth and/or regeneration of the neuron.
• soluble RGMa 1-133 is the most potent inhibitor of axonal growth of the RGMa ligands followed by soluble RGMa ⁇ ee (e.g. 22kDa fragment) (see for example Fig 9).
• soluble RGMa fragments can provide long range signals. SKI-1 and/or furin by inhibiting release of soluble RGMa fragments, can promote long range neuron survival and axon growth. Accordingly, in an embodiment the method is for stimulating long range neuron survival, axon growth and/or regeneration.
• the neural tissue is provided with the SKI-1 inhibitor and/or the furin inhibitor by administering the inhibitor or a composition comprising the inhibitor to the subject by injection into the neural tissue.
• the neural tissue is provided with SKI-1 inhibitor and/or the furin inhibitor by intravenous injection.
• Another aspect includes a method of promoting neuron survival, axon growth and/or regeneration in a subject in need thereof, the method comprising administering to the subject a SKI- 1 and/or Furin inhibitor sufficient to promote growth and/or regeneration of neuron axons.
• Axonal growth and regeneration can be important for improving nerve signal transduction in damaged nerves, damaged for example by trauma and/or disease.
• the central nervous system has limited capacity to repair damaged nerves.
• Nerves comprise bundles of axons from different neurons.
• a neuron consists of a cell body, branch-like extensions called dendrites, and at least one longer extension called an axon.
• the dendrites conduct signals toward the neuron cell body and the axon carries messages away from the cell body toward the terminal end of the axon to communicate with other cells.
• Nerves connect the brain to effector cells. The signal between the cell body and effector cells is interrupted if neurons are severely damaged. Nerve damage can be a result of nerve injuries resulting from nerve trauma such as a spinal cord injury.
• Nerve trauma can for example be incurred through high impact accidents such as motor vehicle accidents, severe falls and lacerations that lead to nerve compression and/or nerve severance.
• damage to the lower spinal cord if severe can lead to paraplegia, paralysis of the lower extremities.
• Severe damage to the upper spinal column may lead to quadriplegia, paralysis of all four extremities.
• the subject has a nerve injury and/or nerve damage.
• a further aspect includes a method of treating nerve damage in a subject in need thereof, the method comprising the step of administering to the subject a SKI-1 and/or Furin inhibitor sufficient to promote regeneration of neuron axons. Also provided is use of a SKI-1 and/or Furin inhibitor for promoting survival and/or regeneration of neuron axons and/or treat nerve damage in a subject in need thereof. In another embodiment, the disclosure includes a SKI-1 and/or Furin inhibitor for use to promote survival and/or regeneration of neuron axons and/or treat nerve damage in a subject in need thereof.
• the nerve injury and/or damage is a spinal cord injury.
• the nerve damage is damage of the optic nerve for example as seen in multiple sclerosis and/or nerve damage in the spine for example as seen in multiple sclerosis.
• the nerve damage is damage of CNS connections following stroke.
• the nerve damage is damage of neuronal networks seen for example in Parkinson disease and Alzheimer's disease.
• Nerve damage can be caused by stroke.
• Zhang et al reported that the levels of RGMa are significantly elevated after ischemia/reperfusion injury in a rodent model.
• Olfactory stimulation treatment downregulated the expression of RGMa, reduced infarct volume and improved neurological function.
• Administration of a SKI-1 inhibitor is demonstrated herein to reduce infarct volume and edema and improve neurological outcome.
• the subject has suffered and/or is suffering a stroke (e.g. ischemic neuronal damage).
• a stroke e.g. ischemic neuronal damage
• An embodiment includes a method of reducing ischemic injury in a subject in need thereof comprising administering a PPC inhibitor, optionally a SKI-1 inhibitor and/or a furin inhibitor to the subject.
• a PPC inhibitor for example a SKI-1 and/or Furin inhibitor to reduce ischemic injury in a subject in need thereof.
• the disclosure includes a PPC inhibitor, for example a SKI-1 and/or Furin inhibitor for use to reduce ischemic injury in a subject in need thereof.
• Another aspect includes a method of treating an ischemic neuronal injury comprising administering to a subject in need thereof a composition comprising a SKI-1 inhibitor and/or a Furin inhibitor.
• a SKI-1 and/or Furin inhibitor to treat ischemic injury in a subject in need thereof.
• the disclosure includes a SKI-1 and/or Furin inhibitor for use to treat ischemic injury in a subject in need thereof.
• Nerve damage can be caused by neurodegenerative diseases including but not limited to multiple sclerosis, Alzheimer's disease, dementia, diabetes, Parkinson's disease and Huntington's disease.
• the need for neuron survival, axonal growth and/or regeneration is a common theme in neurodegenerative disease.
• Another aspect includes a method of treating a neurodegenerative disease, the method comprising administering to a subject in need thereof a PPC inhibitor, optionally a SKI-1 inhibitor and/or a Furin inhibitor, and/or a composition comprising the PPC inhibitor.
• a PPC inhibitor for example a SKI-1 and/or Furin inhibitor treating a neurodegenerative disease in a subject in need thereof.
• the disclosure includes a PPC inhibitor, for example a SKI-1 and/or Furin inhibitor for use in treating a neurodegenerative disease in a subject in need thereof.
• SKI-1 inhibitors are sufficient to prevent the release of soluble RGMa, such as N-RGMa fragments and NN-RGMa.
• administration of a SKI-1 inhibitor significantly reduced the infarct volume and edema compared to Control. Additionally, neurological outcome was markedly improved in animals treated with SKI-1 inhibitor at 48h post-stroke induction.
• inactivation of SKI-1 which prohibits the cleavage of full-length RGMa into its active fragments and thus binding to Neogenin, promotes cell survival and axonal outgrowth.
• the amount administered is for example an effective amount to induce neuron survival, axon growth and/or regeneration.
• RGMa has been implicated in multiple sclerosis. Indeed a genetic link has been suggested between RGMa mutations and the development of the disease in human patients (Nohra et al., 2010) and RGMa has been shown to activate the immune system in experimental models of MS (Muramatsu et al., 2011 ).
• the subject has multiple sclerosis and the method, use or inhibitor is for treating multiple sclerosis.
• Muramatsu et al 2011 also showed neutralizing antibodies to RGMa attenuated clinical symptoms of mouse myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) and reduced invasion of inflammatory cells into the CNS.
• MOG mouse myelin oligodendrocyte glycoprotein
• EAE experimental autoimmune encephalomyelitis
• a further embodiment includes a method of inhibiting an immune response in CNS tissues, e.g. brain and spinal cord in multiple sclerosis patients comprising administering a SKI-1 inhibitor and/or a furin inhibitor.
• Another embodiment includes a method of inhibiting demyelination in a subject in need thereof comprising administering a SKI-1 inhibitor and/or a furin inhibitor.
• Yet another aspect includes a method of treating a subject with a multiple sclerosis comprising administering to a subject in need thereof a PPC inhibitor, optionally a SKI-1 inhibitor and/or a Furin inhibitor, and/or a composition comprising the PPC inhibitor.
• the PPC inhibitor is for treating glaucoma, stroke, MS, Parkinson's disease, Alzheimer's disease, nervous system inflammatory conditions, and/or spinal cord and peripheral nerve injuries in a subject in need thereof.
• administration of a PPC inhibitor significantly reduces tissue damage in a stroke rodent model and improves neurological outcome.
• Treatment efficacy can be monitored for example by conducting neurological tests known in the art, relevant for the particular nerve damage.
• the PPC inhibitor is selected from a serine protease inhibitor, optionally a membrane permeable serine protease inhibitor if the inhibitor is used for applications involving cells.
• the membrane permeable serine protease inhibitor is selected from an AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) peptide and an ER permeable serine protease inhibitor.
• the PPC inhibitor is RVKR peptide (SEQ ID NO:3) for example decanoyl-RVKR-cmk and derivatives.
• the PPC inhibitor is a SKI-1 inhibitor, such as RRLL peptide (SEQ ID NO:4), PF-429242 (Pasquato et al, 2012), peptide CIYISRRLLC (SEQ ID NO:5; optionally with terminal "C” residues cyclized) (Pasquato et al, 2011 ) and/or prosegment inhibitor R134E.
• SKI-1 inhibitor such as RRLL peptide (SEQ ID NO:4), PF-429242 (Pasquato et al, 2012), peptide CIYISRRLLC (SEQ ID NO:5; optionally with terminal "C” residues cyclized) (Pasquato et al, 2011 ) and/or prosegment inhibitor R134E.
• the peptide inhibitors for example RVKR (SEQ ID NO:3), RRLL (SEQ ID NO:4) and CIYISRRLLC (SEQ ID NO:5; optionally with terminal "C” residues cyclized) can be synthesized using methods known in the art .
• the peptides can also be modified for example by addition of N and C term moieties including for example CMK and DecanoyI to promote cell permeability.
• N and C term moieties including for example CMK and DecanoyI to promote cell permeability.
• cyclic peptides can be prepared using methods known in the art
• the PPC inhibitor is a furin inhibitor.
• the furin inhibitor is a furin prosegment inhibitor
• the inhibitors can be suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.
• compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
• Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20 th edition).
• the compositions include, albeit not exclusively, solutions of the substances in association with one or more than one pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
• compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which optionally further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient.
• Other components that are optionally present in such compositions include, for example, water, surfactants (such as TweenTM), alcohols, polyols, glycerin and vegetable oils.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions.
• the composition can be supplied, for example, but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the subject.
• Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition.
• suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1 (2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride (DOT A), diolesyl-phosphotidyl-ethanolamine (DOPE), and liposomes.
• DOT A N-(1 (2,3-dioleyloxy)propyl)N,N,N- trimethylammonium chloride
• DOPE diolesyl-phosphotidyl-ethanolamine
• liposomes Such compositions should contain a therapeutically effective amount of the inhibitor(s) together with a suitable amount of carrier so as to provide the form for direct administration to the subject.
• the inhibitor(s) and/or composition comprising the inhibitor(s) is/are administered, for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.
• the disclosure describes a pharmaceutical composition wherein the dosage form is an injectable dosage form.
• An injectable dosage form is to be understood to refer to liquid dosage forms suitable for, but not limited to, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, or intranasal administration.
• Solutions of compounds described herein can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose.
• a surfactant such as hydroxypropylcellulose.
• a sodium chloride solution for example a 0.9% sodium chloride solution or a dextrose solution for example a 5% dextrose solution.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20 th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
• the inhibitor(s) and/or composition is/are administered by intravenous, intraspinal and/or intracranial infusion. In another embodiment he inhibitor(s) and/or composition is/are administered by Intraperitoneal and/or intrathecal application.
• the PPC inhibitor, optionally SKI-1 inhibitor and/or furin inhibitor is administered in conjunction with another therapy.
• Wang et al reported that olfactory stimulation reduced levels of RGMa in an ischemia/reperfusion injury, a model for stroke, and was associated with improved response.
• the PPC inhibitor optionally a SKI-1 inhibitor and/or furin inhibitor, is administered in conjunction with additional agent.
• additional agent may be administered separately, as part of a multiple dose regimen, from the PPC inhibitor.
• these agents may be part of a single dosage form, mixed together with PPC inhibitors in a single composition.
• these agents can be given as a separate dose that is administered at about the same time.
• kits comprising one or more of a PPC inhibitor, such as a SKI-1 inhibitor and/or a furin inhibitor, a RGMa fragment, a composition, isolated polypeptide, isolated nucleic acid, and/or an antibody, described herein, instructions for use, and/or a vial for housing the PPC inhibitor, RGMa fragment or antibody.
• a PPC inhibitor such as a SKI-1 inhibitor and/or a furin inhibitor
• RGMa fragment a composition, isolated polypeptide, isolated nucleic acid, and/or an antibody, described herein
• instructions for use and/or a vial for housing the PPC inhibitor, RGMa fragment or antibody.
• the RGMa fragment and/or antibody is attached to a substrate.
• the antibody comprises a detectable label, preferably capable of producing, either directly or indirectly, a detectable signal.
• the label may be radio-opaque or a radioisotope, such as 3 H, 4 C, 32 P, 35 S, 23 l, 125 l, 131 l; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion.
• a radioisotope such as 3 H, 4 C, 32 P, 35 S, 23 l, 125 l, 131 l
• a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as fluorescein isothiocyanate, rhodamine or luciferin
• an enzyme such as alkaline phosphatase, beta-galactosidase
• the nervous system is enormously complex, yet the number of cues that control axonal growth is surprisingly meager. Post-translational modifications amplify diversity, but the degree to which they are employed is unclear.
• Furin and SKI-1 combine with autocatalytic-cleavage and a disulphide-bridge to generate four membrane-bound and three soluble forms of the Repulsive Guidance Molecule (RGMa).
• RGMa Repulsive Guidance Molecule
• N- and C-RGMa fragments bound the same Fibronectin-like domains in Neogenin and blocked outgrowth. This represents an example in which unrelated fragments from one molecule inhibit outgrowth through a single receptor-domain.
• RGMa is a tethered membrane-bound molecule, proteolytic processing amplifies RGMa diversity by creating soluble versions with long-range effects as well.
• RGMa is processed into soluble proteins that inhibit axonal growth
• the 60 kDa has been described before and results from RGMa shedding from the membrane (RGMaA) by a phospholipase (Hata et al. , 2006) and may not be biologically relevant as it is not seen in brain matrices.
• the 30 kDa band was broad, so we surmised that it represented a glycosylated N-terminal fragment.
• PNGaseF was removed N-glycosylations using PNGaseF and assessed its resulting molecular mass by Western blot.
• Furin and SKI-1 may regulate the activity of other guidance molecule. Indeed, Furin activates Semaphorins, which are critical for the growth of many axons (Adams et al., 2007). While these experiments do not directly show that RGMa cleavage by Furin and SKI-1 regulates axonal paths, they are consistent with that model and provide in vivo evidence that these enzymes play a critical role in the regulation of axonal growth.
• RGMa cleavage is required for Neogenin binding and outgrowth inhibition
• C- RGMa TEV C- RGMa TEV
• a construct in which the original autocatalytic cleavage site between N- and C-RGMa was replaced by a TEV cleavage site
• RGMa is GPI anchored and has the same action on growing fibers as ephrins (Drescher et al., 1997), it has been assumed that only membrane bound RGMa inhibits axons (Monnier et al., 2002).
• the release of three soluble RGMa proteins (RGMaA, N-RGMa and NN-RGMa; Figure 1 A) has never been reported before, hence their functions remain unknown.
• To assess the functions of soluble forms of RGMa we tested purified proteins on growing fibers (Figure 10). Strikingly, RGMaA, N-RGMa and NN-RGMa significantly inhibited retinal fibers in a concentration-dependent manner ( Figure 9B,C).
• NIE-115 cells expressing a control shRNA extended shorter processes on RGMaA, N-RGMa and NN-RGMa compared to laminin alone, which was suppressed in cells expressing Neogenin-shRNA (Figure 9D,E).
• NN-RGMa (5 ⁇ g/ml) was more potent than N-RGMa (10 ⁇ g/ml) and RGMaA (20 pg/ml; Figure 9E).
• Figure 9C these data show that Neogenin mediates inhibition by N-RGMa, NN-RGMa, and RGMaA ( Figure 9D,E).
• RGMa soluble proteins display various affinities to Neogenin
• N-RGMa and NN-RGMa perturb pathfinding in vivo
• RGMa gain- and loss- of function experiments indicate that it is a key protein for the establishment of visual maps (Matsunaga et al., 2006). However, these experiments did not establish which RGMa fragment(s) is (are) involved in pathfinding. The fact that secreted RGMa proteins may control retinal axon pathfinding was unexpected as RGMa was believed to i) function similar to ephrins, which require polymerization in membranes to guide axons (Egea and Klein, 2007) and ii) use its C-terminal portion to inhibit axonal growth.
• Neogenin domain(s) with which C- and N-RGMa proteins interact The extracellular portion of Neogenin contains four Immunoglobulin like (4lg) and six Fibronectin type III (6FNIII) domains ( Figure 13A). It has been reported that 6FNIII interacts with full length RGMa (Rajagopalan et al., 2004). Using 6FNIII-AP, we showed that C- and N-RGMa proteins interact with 6FNIII ( Figure 13A). Next, we sought to identify which sub-region of the 6FNIII domain(s) interact with N- and C-RGMa. To do so, we generated Neogenin constructs that contain only some of the 6 FN 111 domains ( Figure 13A).
• Retinal axons were grown on RGMa proteins in the presence of I pg/ml of FNII 1(3-4). Remarkably, the presence of this fragment restored axonal growth on RGMaA, N- RGMa, and C-RGMa, indicating that it blocked the inhibitory activities of these proteins on retinal axons ( Figure 13E-F). Together these data represent a unique example in which two unrelated domains of a same guidance molecule inhibit axonal growth through interaction with the same receptor region.
• RGMa has critical roles in axonal growth, cell differentiation, apoptosis, neuronal regeneration, and bone development (Monnier et al., 2002; Matsunaga et al., 2004, 2006; Hata et al., 2006; Zhou et al., 2010).
• RGMa In order to interact with Neogenin, RGMa needs to be cleaved. Because the interaction between RGMa and Neogenin is demonstrated in spinal cord injury and Multiple sclerosis (Hata et al., 2006; Muramatsu et al., 2011 ) it logic to postulate that preventing RGMa cleavage will promote regeneration in spinal cord injuries and treat MS, respectively.
• PCs Proprotein Convertases
• Furin and SKI-1 are involved in RGMa processing and generate N-terminal soluble and C-terrninal membrane bound proteins with different inhibitory activities.
• RGMa fragments with no apparent sequence homology all interacted with the same Fibronectin domains in Neogenin to inhibit axonal growth.
• RGMa and ephrin-A5,-A2 are GPI-anchored proteins that guide retinal fibers (Monnier et al., 2002; Drescher et al., 1997). Thus, they have both been assumed to function as membrane bound cues. As shown here, however, soluble RGMa proteins can also strongly inhibit fiber growth. This is in stark contrast to ephrins, which require oligomerization in membrane clusters to be functionally active (Egea and Klein, 2007). Activity of ephrins is also regulated by proteolytic processing.
• ephrin-A2 is cleaved by Kuzbanian after binding to its receptor, a mechanism that leads to axon detachment and termination of signaling (Hattori et al., 2000). Strikingly, release of RGMa from the membrane has the opposite effect since un-cleaved membrane bound protein is inactive, and proteolysis creates active soluble RGMa proteins.
• this new data invoke a new mechanism of action for RGMa in which the combination of long (soluble) and short (membrane bound) range guidance regulates topographic mapping.
• RGMa represents the first example in which multiple inhibitory fragments from a single ligand regulate axonal growth through the same receptor domain.
• RGMa is involved in neurodegenerative diseases. Thus, it is a major impediment to neuronal regeneration and antibodies that neutralize C-RGMa promote regeneration (Hata et al., 2006). Moreover, there is growing evidence that RGMa is a key player in multiple sclerosis (Muramatsu et al., 2011 ; Nohra et al., 2010). In the light of the new data disclosed herein, multiple RGMa-fragments may contribute to the negative environment that hampers regeneration following CNS injury. Optimal approaches to deactivate RGMa should target all inhibitory C- and N-terminal fragments. The newly generated data indicate that specific SKI-1 inhibition, using for example the RRLL peptide, is sufficient to prevent the formation of C- and N-terminal RGMa peptides (Fig.14).
• C-RGMa-TEV was cloned by inserting a TEV cleavage site between N-RGMa and C- RGMa.
• Membranes were prepared from transfected cells, washed and resuspended in 1x TEV buffer and cleaved with AcTEV protease (Invitrogen) ON at 4°C. Membranes were washed to remove the cleaved N-terminal part, and re-suspended in PBS.
• Soluble proteins were purified using Ni-NTA agarose (Invitrogen), and dialyzed in PBS. Anti-His (Qiagen), and anti RGMa (8B6; Tassew et al., 2009) were used.
• Binding Assay A 96 well plate was coated with i) Poly-L-Lysine (100 ⁇ , 10pg/ml) and ii) membrane suspensions (100 ⁇ ) adjusted to an OD of 0.1 (at 220 nm) were added to the wells. Plates were then centrifuged at 3000rpm (15min at 4oC), blocked with 5% BSA for 1 h, and different concentrations of AP-tagged proteins were added for 3h. Wells were washed with PBS, incubated at 65oC for 1 h to deactivate endogenous AP and developed with 1 mg/ml pNPP.
• Neogenin silencing and neurite outgrowth Mouse Neogenin shRNA and control shRNA were gifts from Dr. Yamashita T. NIE-115 cells which endogenously express Neogenin were co-transfected with shRNA and GFP. 24 h later, cells were plated on coverslips coated with membranes from Mock, wtRGMa, D149A and H151A (OD of 0.1 at 220nm). Alternatively, cells were cultured on molar equivalent amounts of soluble proteins, RGMaA (20 g/ml), N- RGMa ( ⁇ g/mL) and NN-RGMa (5pg/ml). Cells were differentiated in 2% DMSO and neurite length was measured 48h later.
• Explants were fixed in 4%PFA, permeablized with 0.1 %Triton X100, stained with Alexa488-fluor-phalloidin and viewed under a fluorescence microscope (Zeiss). The number and length of fibers were then quantified using Image Pro 5.0. Only explants which displayed growth were considered.
• Viral titer was determined by infecting DF1 cells with serial dilutions and staining for the gag protein (AMV-3C2 Ab; DSHB). Titers of 1x10 8 IU/mL were used for infections.
• Cerebral Focal Ischemia Model Female Sprague-Dawley rats weighing 200-250 g were housed in a 12-h light: 12-h dark cycle and had free access to water and food. Focal cerebral ischemia was induced by injection of a preformed clot into the MCA, the rats was initially anesthetized with 3.0% isoflurane and then maintained with 1.5% isoflurane in a mixture of 30:70 0 2 :N0 2 with a face mask during surgery. Body temperature was maintained at 37°C in the normothermic rats with a heating pad for the duration of surgery and immediate postoperative period until the animal recovered fully from anesthesia. A 1.5-cm longitudinal incision was made in the midline of the ventral cervical skin.
• the right common carotid artery, right internal carotid artery, and right external carotid were exposed.
• the distal portion of the ECA was ligated and cut.
• Ten microliters of blood was withdrawn into the catheter and retained for 15 minutes to allow formation of a clot. Once the clot is formed, the catheter was advanced 17 mm into the internal carotid artery until its tip is 1-2 mm away from the origin of the MCA. The preformed clot in the catheter was then injected, and the catheter removed.
• the infarct volume was expressed as a percentage of the total volume from the ipsilateral hemisphere.
• Brain edema was determined by calculating the volume difference between the 2 hemispheres and dividing by the volume of the left hemisphere.
• 48 hours after MCA occlusion the anesthetized rats was killed.
• the brains was removed from the skull and cooled in ice-cold saline for ⁇ 5 minutes.
• For morphometric examination 2 mm-thick coronal sections were cut using a rat brain matrix. A total of 8 coronal sections was collected, and the sections was stained using a 2% 2, 3, 5-triphenyltetrazolium chloride solution.
• the infarct appears pale white on a background of red normal brain.
• the stained brain sections were scanned and the images were analyzed. Determination of infarct volume and edema in 2 groups were blinded.
• the animals were assessed and assigned a score based on the following grading system: 0, no observable deficit(normal); 1 , forelimb flexion(moderate deficits); 2, forelimb flexion plus decreased resistance to lateral push(moderate deficits); 3, unidirectional circling(severe deficits); and 4, unidirectional circling plus decreased level of consciousness(severe deficits).
• RGMa release was studied after transfection of HEK-293 cells with full length RGMa.
• RGMa delta two RGMa peptides are apparent -soluble full length RGMa (RGMa delta) plus N-RGMa peptides.
• RGMa delta apparent -soluble full length RGMa
• N-RGMa peptide In the presence of either RRLL Or RVKR, none of the N-RGMa peptide is apparent ( Figure 14) indicating that SKI-1 inhibition is sufficient to block release of N-RGMa peptides.
• Example 3 [00243] Hata et al 2006 teach that a domain important for functional activity in chick RGM is the COOH-terminal 150-200 amino acids of the active RGM protein. A synthetic peptide (residues 309-322) was selected as immunogen to generate anti-rat RGMa rabbit antisera. The Hata experiments suggest that neutralizing C-RGMa promotes regeneration. The present data show that not only C-RGMa inhibits regeneration but also that N-RGMa is involved and even more potent. To neutralize both activities, inhibitors of SKI-1 that prevent activation into C- and N-terminal fragments are proposed.
• RGMa is upregulated following stroke. RGMa may therefore be involved in the non-regenerative property of the CNS.
• Schwab et al., 2005 reported following central nervous system injury, RGM, a novel, potent axonal growth inhibitor, is present in axonal growth impediments: the mature myelin, choroid plexus, and components of the developing scar.
• amino acids 1-46 can be present or absent; amino acid 168 can be D or A; amino acid 170 can be H or A
• SEQ ID NO: 9 (includes tev)
• amino acids 1-46 can be present or absent; amino acid 168 can be D or A; amino acid 170 can be H or A
• proprotein convertase PC5A and a metalloprotease are involved in the proteolytic processing of the neural adhesion molecule L1. J. Biol. Chem. 278, 10381-8.
• RGM is a repulsive guidance molecule for retinal axons. Nature 419, 392-395.

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