Jnk3 modulators and methods of use

Abstract

The c-Jun NH 2 -terminal kinase (JNK) group of MAP kinases are activated by exposure of cells to environmental stress. The role of JNK in the brain was examined by targeted disruption of the gene that encodes the neuronal isoform JNK3. It was found that JNK3 is required for the normal response to seizure activity. Methods of screening for molecules and compounds that decrease JNK3 expression or activity are described. Such molecules or compounds are useful for treating disorders involving excitotoxicity such as seizure disorders, Alzheimer's disease, Huntington disease, Parkinson's disease, and ischaemia.

1 . A method of identifying a compound that modulates JNK3 expression, the method comprising:
 incubating a cell that can express a JNK3 protein with a compound under conditions and for a time sufficient for the cell to express a JNK3 protein absent the compound;   incubating a control cell under the same conditions and for the same time absent the compound;   measuring JNK3 expression in the cell in the presence of the compound;   measuring JNK3 expression in the control cell; and   comparing the amount of JNK3 expression in the presence and absence of the compound,   
       wherein a difference in the level of expression indicates that the compound modulates JNK3 expression. 
     
     
         2 . The method of  claim 1 , wherein the compound decreases the expression of JNK3. 
     
     
         3 . A method of identifying a compound that modulates JNK3 activity, the method comprising:
 incubating a cell that has JNK3 activity with a compound under conditions and for a time sufficient for the cell to express JNK3 activity absent the compound;   incubating a control cell under the same conditions and for the same time absent the compound;   measuring JNK3 activity in the cell in the presence of the compound;   measuring JNK3 activity in the control cell; and comparing the amount of JNK3 activity in the presence and absence of the compound,   
       wherein a difference in the level of activity indicates that the compound modulates JNK3 activity. 
     
     
         4 . The method of  claim 3 , wherein the compound decreases JNK3 activity. 
     
     
         5 . A method of identifying a compound that modulates the binding of a JNK3 polypeptide to a substrate, said method comprising comparing the amount of a JNK3 polypeptide bound to a substrate in the presence and absence of a selected compound, wherein a difference in the amount of binding of a JNK3 polypeptide to a substrate indicates that said selected compound modulates the binding of a JNK3 polypeptide. 
     
     
         6 . The method of  claim 5 , wherein the binding of a JNK3 polypeptide to a substrate is decreased. 
     
     
         7 . A method for generating a totipotent mouse cell comprising at least one inactivated JNK3 gene, the method comprising:
 a. providing a plurality of totipotent mouse cells;   b. introducing into the cells a DNA construct comprising a disrupted mouse JNK3 gene, wherein the JNK3 gene is disrupted by insertion of a nucleotide sequence into the gene that prevents expression of functional JNK3;   c. incubating the cells such that homologous recombination occurs between the chromosomal sequence encoding JNK3 and the introduced DNA construct; and   d. identifying a totipotent mouse cell comprising at least one inactivated JNK gene.   
     
     
         8 . A method for generating a mouse homozygous for an inactivated JNK3 gene comprising:
 a. providing a totipotent mouse cell comprising at least one inactivated JNK3 gene;   b. inserting the cell into a mouse embryo and implanting the embryo into a female mouse;   c. permitting the embryo to develop into a neonatal mouse;   d. permitting the neonatal mouse to reach sexual maturity; and   e. mating two sexually mature mice of step d. to obtain a mouse homozygous for the inactivated JNK3 gene(−/−), wherein the homozygous JNK3(−/−) mouse is resistant to excitotoxic damage.   
     
     
         9 . A method of treating a patient having or at risk for a disorder involving excitotoxicity, the method comprising administering to the patient a therapeutically effective amount of a compound that inhibits JNK3 expression. 
     
     
         10 . The method of  claim 9 , wherein the compound is an antisense nucleic acid molecule. 
     
     
         11 . The method of  claim 9 , wherein the disorder is selected from the group consisting of Alzheimer's disease, Huntington disease, ischemia, amyotrophic lateral sclerosis, trauma, motorneuron disease, Parkinson's disease, or epilepsy. 
     
     
         12 . A transgenic non-human mammal having a transgene disrupting expression of a JNK3 gene, the transgene being chromosomally integrated into germ cells of the mammal. 
     
     
         13 . The mammal of  claim 12 , wherein the mammal is a mouse. 
     
     
         14 . The mammal of  claim 12 , wherein the germ cells are homozygous for the transgene. 
     
     
         15 . The mammal of  claim 12 , wherein the disruption results in a null mutation. 
     
     
         16 . A cell line descended from a cell of the mammal of  claim 12 . 
     
     
         17 . A DNA construct comprising a disrupted mouse JNK3 gene, including an insertion of a nucleotide sequence into the gene that prevents or modifies the expression of functional JNK3.