Co-factors for trophic factors, and methods of use, thereof

Abstract

The present invention is based on the discovery and isolation of a co-factor for trophic factors. It has been discovered that trophic factors require a co-factor to stimulate and/or potentiate the trophic factor activity and/or specificity. This was clearly identified in low density cells where trophic factors are unable, or at best, at minimal levels, able to proliferate undifferentiated cells without a co-factor. In a particular embodiment of the present invention, there is provided a composition comprising glycosylated cystatin C (CCg), an FGF co-factor that stimulates proliferation of neural and fibroblast associated undifferentiated cells. The N-glycosylation of cystatin C is required for its activity. Moreover, CCg acts in cooperation with basic fibroblast growth factor (FGF-2) to induce neural progenitor cell proliferation.

What is claimed is:  
     
         1 . Substantially purified glycosylated cystatin C (CCg) polypeptide, or a biologically active fragment thereof  
     
     
         2 . The glycosylated cystatin C polypeptide of  claim 1 , wherein said polypeptide comprises about fifteen hexose residues.  
     
     
         3 . The glycosylated cystatin C polypeptide of  claim 2 , wherein said glycosylated cystatin C polypeptide further comprises a terminal sialic acid linked to a terminal hexose residue.  
     
     
         4 . A method for inhibiting the activity and/or specificity of trophic factors, said method comprising contacting a cell with an inactivated co-factor for said trophic factor.  
     
     
         5 . The method of  claim 4 , wherein said method inhibits proliferation of a mammalian neural cell, comprising: 
 contacting said cell with a non-glycosylated form of cystatin C under conditions that inhibit said cell from proliferating.    
     
     
         6 . The method of  claim 5 , wherein said mammalian neural cell is a human cell.  
     
     
         7 . The method of  claim 4 , wherein said method inhibits FGF mediated processes, comprising: 
 contacting said cell with a non-glycosylated form of cystatin C under conditions that inhibit FGF activity and/or specificity.    
     
     
         8 . A method of identifying a peptide which inhibits neural cell proliferation comprising: 
 contacting a neural progenitor cell with a peptide fragment of non-glycosylated cystatin C and glycosylated cystatin C; and    comparing the ability of said neural progenitor cell to proliferate in the presence of the peptide fragment with to the ability of a neural progenitor cell to proliferate in the absence of said peptide fragment.    
     
     
         9 . The method of  claim 8 , further comprising contacting the cell with fibroblast growth factor-2 prior to comparing the ability of said neural progenitor cell to proliferate in the presence of the peptide fragment with to the ability of a neural progenitor cell to proliferate in the absence of the peptide fragment.  
     
     
         10 . A method of identifying an agent which stimulates and/or potentiates trophic factor activity and/or specificity comprising: 
 contacting cell in low density with a trophi factor and a test agent under conditions that allow the components to interact; and    comparing the ability of said cells to proliferate in the presence of the agent to the ability of the cells to proliferate in the absence of said agent.    
     
     
         11 . The method of identifying an agent which promotes neural cell proliferation comprising: 
 contacting a neural progenitor cell with a test agent and with glycosylated cystatin C under conditions that allow the components to interact; and    comparing the ability of said neural progenitor cell to proliferate in the presence of the agent to the ability of a neural progenitor cell, to proliferate in the absence of said agent.    
     
     
         12 . A method for ameliorating a neural disorder in a subject having such a disorder, comprising: 
 administering a therapeutically effective amount of neurotrophic co-factor to said subject, thereby ameliorating the disorder.    
     
     
         13 . The method of  claim 12 , wherein said co-factor is glycosylated cystatin C.  
     
     
         14 . The method of  claim 12 , wherein said disorder is selected from the group consisting of Alzheimer's disease, Parkinson's disease, and stroke.  
     
     
         15 . A pharmaceutical composition comprising a therapeutically effective amount of a co-factor for trophic factors and a pharmaceutically acceptable carrier.  
     
     
         16 . The pharmaceutical composition of  claim 15 , further comprising said trophic factor.  
     
     
         17 . An antibody which binds glycosylated cystatin C with a different affinity than non-glycosylated stem cell glycocystatin C.  
     
     
         18 . A method for identifying co-factors for a trophic factor, comprising 
 culturing cells in low density with a conditioned medium and said trophic factor,    fractionating said condition medium, and    purifying said co-factor which proliferates said cells.    
     
     
         19 . The method of  claim 18 , wherein said conditioned media is autocrine/paracrine.  
     
     
         20 . The method of  claim 18 , wherein said cells are undifferentiated.  
     
     
         21 . The method of  claim 18 , wherein said trophic factor is a neurotrophin.  
     
     
         22 . The method of  claim 18 , wherein said fractionation comprises multiple fractionations.  
     
     
         23 . A co-factor produced from the method of  claim 18 .  
     
     
         24 . Substantially purified stem cell glycocystatin C polypeptide, wherein said polypeptide is produced by a method comprising: 
 obtaining conditioned media from a mammalian neural progenitor cell;    performing affinity chromatography on said media and eluting a fraction having mitogenic activity;    performing papain chromatography on said fraction having mitogenic activity; and    recovering a polypeptide of about 21 kDa.    
     
     
         25 . The polypeptide of  claim 24 , wherein said neural progenitor cell is a hippocampus-derived neural progenitor cell.  
     
     
         26 . The polypeptide of  claim 15 , wherein said mammalian progenitor cell is selected from the group consisting of a human, a rat, a mouse, a bovine, and an ovine neural progenitor cell.  
     
     
         27 . A method for purifying stem cell glycocystatin C polypeptide, comprising: 
 obtaining conditioned media from a neural progenitor cell;    performing affinity chromatography on said media and eluting a fraction having mitogenic activity;    performing papain chromatography on said faction having mitogenic activity; and    recovering a polypeptide of about 21 kDa.    
     
     
         28 . An assay system for identifying a co-factor of FGF when the system is contacted with a putative co-factor comprising: 
 culturing multi-potent mammalian neural progenitor cells at a low density in a basal medium comprising FGF.    
     
     
         29 . The assay system of  claim 28 , wherein said multipotent neural progenitor cells are adult neural stem cells.  
     
     
         30 . The assay system of  claim 29 , wherein said adult neural progenitor cells are adult hippocampus-derived neural stem cell.  
     
     
         31 . A co-factor polypeptide, wherein said co-factor is required by trophic factors for activity and/or specificity.  
     
     
         32 . The co-factor of  claim 31 , wherein said co-factor is an autocrine/paracrine co-factor.