Methods for Assessing CDK5 Activation and Function

Abstract

As described herein, signaling events occurring in neurons or at neuronal synapses have been identified that involve Cdk5 and various other molecules which bind to, are activated by, and/or activate Cdk5. Of particular relevance are interactions that stimulate calpain cleavage of p35 into p25, which binds Cdk5 in pathologic states. Assays to identify modulators of these interactions are provided.

What is claimed: 
     
         1 . A method of identifying an inhibitor of Cdk5/p25 complex formation comprising:
 (a) providing a system that permits formation of Cdk5/p25 complex formation;   (b) introducing into said system a candidate substance; and   (c) assessing Cdk5/p25 complex formation using a p25 selective antibody,   wherein a candidate substance that reduces Cdk5/p25 complex formation, as compared to Cdk5/p25 complex formation observed in the absence of said candidate substance, identifies said candidate substance as an inhibitor of Cdk5/p25 formation.   
     
     
         2 . The method of  claim 1 , wherein said system is a cell-free system. 
     
     
         3 . The method of  claim 1 , wherein said system is a cell-based system. 
     
     
         4 . The method of  claim 2  or  3 , wherein said cell-free system is derived from, or said cell-based system is based on, a cell selected from the group consisting of continuously dividing cells in culture, primary cultures of neurons derived from animal brain tissue, or neurons either acutely dissociated or occurring in intact brain tissue. 
     
     
         5 . The method of  claim 1 , wherein assessing comprises Western blot or ELISA. 
     
     
         6 . A method of identifying an inhibitor of calpain-dependent p25 formation comprising:
 (a) providing a system that permits cleavage of p35 into p25 by calpain;   (b) introducing into said system a candidate substance; and   (c) assessing p25 formation using a p25 selective antibody,   wherein a candidate substance that reduces p25 formation, as compared to p25 formation observed in the absence of said candidate substance, identifies said candidate substance as an inhibitor of calpain-dependent p25 formation.   
     
     
         7 . The method of  claim 6 , wherein said system is a cell-free system. 
     
     
         8 . The method of  claim 6 , wherein said system is a cell-based system. 
     
     
         9 . The method of  claim 7  or  8 , wherein said cell-free system is derived from, or said cell-based system is based on a cell selected from the group consisting of continuously dividing cells in culture, primary cultures of neurons derived from animal brain tissue, or neurons either acutely dissociated or occurring in intact brain tissue. 
     
     
         10 . The method of  claim 6 , wherein assessing comprises Western blot or ELISA. 
     
     
         11 . A method of identifying an inhibitor of calpain activation comprising:
 (a) providing a system comprising calpain and Cdk5;   (b) introducing into said system a candidate substance; and   (c) assessing Cdk5/caplain complex formation or calpain proteolytic activity,   wherein a candidate substance that reduces Cdk5/calpain complex formation or calpain proteolytic activity, as compared to Cdk5/calpain complex formation or calpain proteolytic activity observed in the absence of said candidate substance, identifies said candidate substance as an inhibitor of calpain activation.   
     
     
         12 . The method of  claim 11 , wherein said system is a cell-free system. 
     
     
         13 . The method of  claim 11 , wherein said system is a cell-based system. 
     
     
         14 . The method of  claim 12  or  13 , wherein said cell-free system is derived from, or said cell-based system is based on a cell selected from the group consisting of continuously dividing cells in culture, primary cultures of neurons derived from animal brain tissue, or neurons either acutely dissociated or occurring in intact brain tissue. 
     
     
         15 . The method of  claim 11 , wherein assessing comprises (a) measuring Cdk5 binding to calpain; or (b) measuring calpain proteolytic activity. 
     
     
         16 . The method of  claim 15 (a), wherein measuring binding comprises gel mobility shift, FRET, BRET, Western blot, protein pull-down, or ELISA-based detection of complex formation. 
     
     
         17 . The method of  claim 15 (b), wherein proteolytic activity is measured by assessing calpain cleavage of spectrin, NR2B, Jun, or PSD-95. 
     
     
         18 . The method of  claim 17 , wherein measuring proteolytic activity comprises Western blotting or ELISA for the reduction in a calpain substrate, or the formation of cleaved forms of a calpain substrate. 
     
     
         19 . A method of identifying an inhibitor of Cdk5-NR2B complex formation comprising:
 (a) providing a system comprising NR2B and Cdk5;   (b) introducing into said system a candidate substance; and   (c) assessing Cdk5/NR2B complex formation or Cdk5 phosphorylation activity,   wherein a candidate substance that reduces Cdk5/NR2B complex formation or reduces Cdk5 phosphorylation activity, as compared to Cdk5/NR2B complex formation or reduces Cdk5 phosphorylation activity observed in the absence of said candidate substance, identifies said candidate substance as an inhibitor of Cdk5-NR2B formation or reduces Cdk5 phosphorylation activity.   
     
     
         20 . The method of  claim 19 , wherein said system is a cell-free system. 
     
     
         21 . The method of  claim 19 , wherein said system is a cell-based system. 
     
     
         22 . The method of  claim 20  or  21 , wherein said cell-free system is derived from, or said cell-based system is based on a cell selected from the group consisting of continuously dividing cells in culture, primary cultures of neurons derived from animal brain tissue, or neurons either acutely dissociated or occurring in intact brain tissue. 
     
     
         23 . The method of  claim 19 , wherein measuring binding comprises gel mobility shift, FRET, BRET, Western blot, protein pull-down, or ELISA-based detection of complex formation. 
     
     
         24 . The method of  claim 19 , wherein assessing comprises (a) measuring Cdk5/NR2B complex formation; or (b) measuring Cdk5 phosphorylation activity.