Methods for shp1 mediated neuroprotection

Abstract

The effect of EPO on the phosphorylation of the EPO receptor, the activation of the MAP Kinase pathway, and the expression of SHP-1 were analyzed. EPO was observed to cause a decrease in the expression of its negative regulator SHP-1. The decrease observed at both the mRNA and protein level was dose dependent and persisted as long as 24 hr following EPO treatment. EPO can down regulate the expression of its own negative regulator as a means for increased potency in neurons. Assays were generated to identify compounds that are useful in regulating SHP1 activity in neural cells.

1 . A method of identifying a compound useful for treating a nervous system condition related to EPOR, comprising the steps of: 1) contacting a test compound with a SHP1 protein or an active fragment thereof and 2) determining the ability of the test compound to decrease the tyrosine phosphatase activity of SHP 1.  
   
   
       2 . The method of  claim 1 , further comprising the steps of a) contacting a neuronal cell with the test compound; b) inducing neurotoxicity in the neuronal cell; c) assaying the cell survival rate in the presence of the test compound, and comparing the cell survival rate with that of a control, wherein the neuronal cell is not treated with the test compound.  
   
   
       3 . The method of  claim 1 , wherein said SHP1 protein or active fragment thereof is substantially purified.  
   
   
       4 . The method of  claim 1 , wherein SHP1 protein or active fragment thereof is expressed from a host cell.  
   
   
       5 . A method of identifying a compound useful for treating a nervous system condition related to EPOR, comprising the steps of: c) contacting a test compound with a regulatory sequence for a SHP1 gene or a cellular component that binds to the regulatory sequence for a SHP1 gene; and d) determining whether the test compound decreases the expression of a gene controlled by said regulatory sequence.  
   
   
       6 . The method of  claim 5 , further comprising the steps of a) contacting a neuronal cell with the test compound; b) inducing neurotoxicity in the neuronal cell; c) assaying the cell survival rate in the presence of the test compound, and comparing the cell survival rate with that of a control, wherein the neuronal cell is not treated with the test compound.  
   
   
       7 . The method of  claim 5 , wherein the gene controlled by the SHP1 regulatory sequence is a reporter gene.  
   
   
       8 . The method of  claim 5 , wherein the gene controlled by the SHP1 regulatory sequence is an SHP1 gene.  
   
   
       9 . The method of identifying a compound useful for treating a nervous system condition related to EPOR, comprising the steps of: e) combining a test compound, a labeled ligand for a SHP1 protein, and a SHP1 protein or an active fragment thereof; and f) measuring the binding of the test compound to the SHP1 protein or active fragment thereof by a reduction in the amount of labeled ligand binding to the SHP1 protein or active fragment thereof.  
   
   
       10 . The method of  claim 9 , further comprising the steps of a) contacting a neuronal cell with the test compound; b) inducing neurotoxicity in the neuronal cell; c) assaying the cell survival rate in the presence of the test compound, and comparing the cell survival rate with that of a control, wherein the neuronal cell is not treated with the test compound.