Prosaposin as a neurotrophic factor

Abstract

Prosaposin, saposin C and various peptide fragments of saposin C stimulate neurite outgrowth in vitro. In addition, prosaposin and saposin C promote increased myelination ex vivo. Prosaposin is present in large neurons of the brain, including both upper and lower motor neurons.

1 . A method for stimulating neural cell outgrowth or increased myelination, comprising:
 contacting neuronal cells with a composition comprising prosaposin or a fragment thereof having the ability to promote increased neural outgrowth or increased myelination activity.   
     
     
         2 . The method of  claim 1  wherein said prosaposin is native. 
     
     
         3 . The method of  claim 1  wherein said prosaposin is recombinantly produced. 
     
     
         4 . The method of  claim 1  wherein said fragment is saposin C. 
     
     
         5 . The method of  claim 1  wherein said fragment is a peptide comprising amino acids 8-29 of saposin C. 
     
     
         6 . The method of  claim 5  wherein said fragment consists essentially of the active neurotrophic fragment located within amino acids 8-29 of SEQ ID NO: 1. 
     
     
         7 . The method of  claim 1  wherein said neuronal cells are neuroblastoma cells. 
     
     
         8 . The method of  claim 7  wherein said neuroblastoma cells are selected from the group consisting of: NS20Y, Neuro 2A and NIE 115 cells. 
     
     
         9 . The method of  claim 1  wherein said neuronal cells are contacted in vitro. 
     
     
         10 . The method of  claim 1  wherein said neuronal cells are contacted in vivo. 
     
     
         11 . The method of  claim 1  wherein said cells are from mouse cerebellar explants. 
     
     
         12 . A method for treatment of demyelination disorders in a mammal comprising: identifying a mammal afflicted with said disorder; and administering to said mammal a pharmaceutically effective demyelination inhibiting amount of prosaposin or a neurotrophic fragment thereof. 
     
     
         13 . The method of  claim 12  wherein said fragment comprises saposin C. 
     
     
         14 . The method of  claim 12  wherein said demyelination disorder is selected from the group consisting of: multiple sclerosis, acute disseminated leukoencephalitis, progressive multifocal leukoencephalitis and adrenal leukodystrophy. 
     
     
         15 . The method of  claim 12  wherein said administration is selected from the group consisting of: intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebrospinal and topical. 
     
     
         16 . The method of  claim 12  wherein said prosaposin or fragment thereof is administered in a biologically compatible carrier. 
     
     
         17 . The method of  claim 12  wherein said prosaposin or fragment thereof is enclosed in a lamellar structure. 
     
     
         18 . A method for halting or slowing the progress of neural or myelin degeneration in neural tissue, comprising: contacting neuronal tissue susceptible to such degradation with prosaposin or an active degradation-inhibiting fragment thereof. 
     
     
         19 . The method of  claim 18  wherein said fragment is saposin C. 
     
     
         20 . The method of  claim 18  wherein said tissue is in vitro. 
     
     
         21 . The method of  claim 18  wherein said tissue is in vivo. 
     
     
         22 . A method for the treatment of neuronal degenerative diseases of the central or peripheral nervous system, comprising administering to a mammal suffering from said disease an amount of a prosaposin fragment effective to retard or halt neuronal degeneration, wherein said fragment includes the neurotrophic activity of the peptide of SEQ ID NO: 1. 
     
     
         23 . The method of  claim 22  wherein said administration is selected from the group consisting of: intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebrospinal, topical and oral. 
     
     
         24 . The method of  claim 22  wherein said disease is a disease of the central nervous system and said fragment is selected to cross the blood brain barrier. 
     
     
         25 . The method of  claim 24  wherein said disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, stroke, post-polio syndrome and amyotrophic lateral sclerosis. 
     
     
         26 . A method for retarding the progress of retinal neuropathy in a patient by administering to the patient an effective amount of prosaposin or a neurotrophic fragment thereof. 
     
     
         27 . The method of  claim 26  wherein said retinal neuropathy is macular degeneration and said patient is a human over the age of 65. 
     
     
         28 . The method of  claim 26  wherein said administration is selected from the group consisting of: topical intravenous, intraocular and oral. 
     
     
         29 . A pharmaceutical composition comprising prosaposin or a neurotrophic fragment thereof in unit dosage form. 
     
     
         30 . A pharmaceutical composition comprising prosaposin or a neurotrophic fragment thereof formulated with a controlled release material. 
     
     
         31 . A neural prosaposin receptor protein in isolated or purified form. 
     
     
         32 . The receptor protein of  claim 31  wherein said receptor is isolated from a P100 plasma membrane fraction by affinity purification using a neurite growth-inducing peptide contained within the saposin C sequence linked to a solid support. 
     
     
         33 . The receptor protein of  claim 31  wherein said receptor has a molecular weight of approximately 20 kDa. 
     
     
         34 . The method of  claim 22  wherein the prosaposin fragment is a peptide comprising amino acids 18-22 of saposin C. 
     
     
         35 . A neurotrophic peptide comprising amino acids 18-22 (Leu-Ile-Asp-Asn-Asn) of SEQ ID NO:3. 
     
     
         36 . A composition comprising the neurotrophic peptide of  claim 35  and a pharmaceutically acceptable carrier. 
     
     
         37 . A composition comprising the neurotrophic peptide of  claim 35 , formulated with a controlled release material. 
     
     
         38 . A composition comprising the neurotrophic peptide of  claim 35 , formulated in lyophilized form. 
     
     
         39 . A composition comprising the neurotrophic peptide of  claim 35 , in liposomal form. 
     
     
         40 . A composition comprising the neurotrophic peptide of  claim 35 , in a form suitable for topical administration. 
     
     
         41 . A composition comprising the neurotrophic peptide of  claim 35 , formulated in unit dosage form. 
     
     
         42 . A composition comprising the neurotrophic peptide of  claim 35 , formulated in a septum sealed vial. 
     
     
         43 . A neurotrophic peptide consisting essentially of amino acids 18-22 (Leu-Ile-Asp-Asn-Asn) of SEQ ID NO:3. 
     
     
         44 . A composition comprising the neurotrophic peptide of  claim 43  and a pharmaceutically acceptable carrier. 
     
     
         45 . A composition comprising the neurotrophic peptide of  claim 43 , formulated with a controlled release material. 
     
     
         46 . A composition comprising the neurotrophic peptide of  claim 43 , formulated in lyophilized form. 
     
     
         47 . A composition comprising the neurotrophic peptide of  claim 43 , in liposomal form. 
     
     
         48 . A composition comprising the neurotrophic peptide of  claim 43 , in a form suitable for topical administration. 
     
     
         49 . A composition comprising the neurotrophic peptide of  claim 43 , formulated in unit dosage form. 
     
     
         50 . A composition comprising the neurotrophic peptide of  claim 43 , formulated in a septum sealed vial. 
     
     
         51 . A method of stimulating neural cell outgrowth or increased myelination, comprising:
 contacting neuronal cells with a composition comprising an effective neurotigenic concentration of a neurotrophic peptide comprising amino acids 18-22 of SEQ ID NO:3.   
     
     
         52 . The method of  claim 51 , wherein the contacting of neuronal cells stimulates neural cell outgrowth. 
     
     
         53 . The method of  claim 51 , wherein the contacting of neuronal cells stimulates increased myelination. 
     
     
         54 . The method of  claim 51 , wherein the neural cell is a peripheral nerve cell. 
     
     
         55 . A method of stimulating neural cell outgrowth or increased myelination, comprising:
 administering to a vertebrate a composition comprising an effective neurotigenic concentration of a neurotrophic peptide comprising amino acids 18-22 of SEQ ID NO:3.   
     
     
         56 . The method of  claim 55 , wherein the administration stimulates neural cell outgrowth. 
     
     
         57 . The method of  claim 55 , wherein the administration stimulates increased myelination. 
     
     
         58 . The method of  claim 55 , wherein the neural cell is a peripheral nerve cell. 
     
     
         59 . The method of  claim 55 , wherein the administration is selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebrospinal, topical and oral. 
     
     
         60 . A method of treating demyelination in a mammal afflicted with demyelination comprising:
 administering to the mammal an effective demyelination inhibiting amount of a neurotrophic peptide comprising amino acids 18-22 of SEQ ID NO:3.   
     
     
         61 . The method of  claim 60 , wherein the demyelination is due to multiple sclerosis, ischemic injury or traumatic injury. 
     
     
         62 . The method of  claim 60 , wherein the demyelination is demyelination of a peripheral nerve cell. 
     
     
         63 . The method of  claim 60 , wherein the administration is selected from the group consisting of intravenous, intramuscular, intradermal, subcutaneous, intracranial, intracerebrospinal, topical and oral. 
     
     
         64 . A method for increasing the survival of retina in a patient in need thereof comprising the step of administering to the patient an amount of a neurotrophic peptide comprising amino acids 18-22 of SEQ ID NO:3, wherein said amount is sufficient to increase the survival of the retina. 
     
     
         65 . The method of  claim 64 , wherein said patient is a human over the age of 65. 
     
     
         66 . The method of  claim 64 , wherein said administering step is selected from the group consisting of topical, intravenous, intraocular and oral. 
     
     
         67 . The method of  claim 64 , wherein the retina has macular degeneration. 
     
     
         68 . A substantially pure polypeptide consisting of the sequence: Tyr-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu (SEQ ID NO:5). 
     
     
         69 . A pharmaceutical composition comprising a neurotrophic polypeptide consisting of amino acids of SEQ ID NO:5 and a pharmaceutically acceptable carrier. 
     
     
         70 . The pharmaceutical composition of  claim 69  which is formulated for containment in a septum sealed vial. 
     
     
         71 . The pharmaceutical composition of  claim 69  which is formulated with a controlled release material. 
     
     
         72 . The pharmaceutical composition of  claim 69  which is formulated in a liposomal form. 
     
     
         73 . A method for treatment of a neurodegenerative disease of the peripheral nervous system which comprises administering to a mammal suffering from said neurodegenerative disease an effective amount of prosaposin or a neurotrophic fragment thereof, wherein said neurotrophic fragment comprises saposin C, a peptide comprising amino acids 8-29 of saposin C, or said fragment includes the active neurotrophic fragment located within amino acids 8-29 of SEQ ID NO:3. 
     
     
         74 . The method of  claim 73 , wherein the neurotrophic fragment is saposin C. 
     
     
         75 . The method of  claim 73 , wherein the neurotrophic fragment is a peptide comprising amino acids 8-29 of saposin C. 
     
     
         76 . The method of  claim 73 , wherein the neurotrophic fragment consists essentially of the active neurotrophic fragment located within amino acids 8-29 of saposin C. 
     
     
         77 . The method of  claim 73 , wherein the neurotrophic fragment is a peptide comprising amino acids 18-22 of saposin C. 
     
     
         78 . The method of  claim 73 , wherein the neurotrophic fragment is a polypeptide consisting of the sequence: Cys-Glu-Phe-Leu-Val-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu (SEQ ID NO:1). 
     
     
         79 . The method of  claim 73 , wherein the neurodegenerative disease of the peripheral nervous system is a peripheral neuropathy. 
     
     
         80 . The method of  claim 79 , wherein the peripheral neuropathy results from diabetes. 
     
     
         81 . The method of  claim 79 , wherein the peripheral neuropathy results from chemotherapy. 
     
     
         82 . A method for treatment of a neurodegenerative disease of the peripheral nervous system which comprises administering to a mammal suffering from said neurodegenerative disease an effective amount of a neurotrophic fragment derived from prosaposin, saposin C, or a peptide comprising amino acids 8-29 of saposin C, wherein said neurotrophic fragment includes the active neurotrophic fragment located within amino acids 8-29 of SEQ ID NO:3. 
     
     
         83 . The method of  claim 82 , wherein the neurotrophic fragment is a polypeptide comprising the sequence: Tyr-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu (SEQ ID NO: 5). 
     
     
         84 . The method of  claim 82 , wherein the neurotrophic fragment is a polypeptide consisting of the sequence: Tyr-Lys-Glu-Val-Thr-Lys-Leu-Ile-Asp-Asn-Asn-Lys-Thr-Glu-Lys-Glu-Ile-Leu (SEQ ID NO:5). 
     
     
         85 . The method of  claim 82 , wherein the neurodegenerative disease of the peripheral nervous system is a peripheral neuropathy. 
     
     
         86 . The method of  claim 85 , wherein the peripheral neuropathy results from diabetes. 
     
     
         87 . The method of  claim 85 , wherein the peripheral neuropathy results from chemotherapy. 
     
     
         88 . A method for treatment of a neurodegenerative disease of the peripheral nervous system which comprises administering to a mammal suffering from said neurodegenerative disease an effective amount of a neurotrophic fragment derived from prosaposin, wherein said neurotrophic fragment comprises amino acids 18-22 of SEQ ID NO:3. 
     
     
         89 . The method of  claim 88 , wherein the neurodegenerative disease of the peripheral nervous system is a peripheral neuropathy. 
     
     
         90 . The method of  claim 89 , wherein the peripheral neuropathy results from diabetes. 
     
     
         91 . The method of  claim 89 , wherein the peripheral neuropathy results from chemotherapy. 
     
     
         92 . A method for treatment of a neurodegenerative disease of the peripheral nervous system which comprises administering to a mammal suffering from said neurodegenerative disease an effective amount of a neurotrophic fragment of prosaposin, wherein said neurotrophic fragment comprises amino acids 18-22 of SEQ ID NO:3. 
     
     
         93 . The method of  claim 92 , wherein the neurodegenerative disease of the peripheral nervous system is a peripheral neuropathy. 
     
     
         94 . The method of  claim 93 , wherein the peripheral neuropathy results from diabetes. 
     
     
         95 . The method of  claim 93 , wherein the peripheral neuropathy results from chemotherapy.