The aim of this proposal is to test strategies for gene therapy in vivo. This will be accomplished by engineering into the mouse genome mutations in the two major excitatory receptor systems in the mammalian brain which mediate fast synaptic transmission, the glutamate receptor and the cholinergic nicotinic receptor system. The glutamate receptor system mediates the vast majority of fast synaptic transmission in the mammalian brain and is implicated by many experiments in memory formation and neurodegenerative disease as is observed in Alzheimer~s patients. The cholinergic nicotinic receptor system has been implicated in memory function and the number of nicotinic receptors is dramatically reduced in Alzheimer~s disease. The hypothesis that a defect in the nicotinic receptor system contributes to the memory deficit observed in Alzheimer~s patients will be tested. Gene therapy strategies will then be attempted in the mutant mice to restore gene expression and cognitive function. The experiments will make use of recombinant DNA technology and the production of mutant mice by genetic engineering methods. Gene targeting methods will be used to knockout or disrupt glutamate receptor and nicotinic receptor genes in the adult mouse using the recently developed CRE/LoxP system. Mutations will be introduced into glutamate receptor genes which alter the glutamate receptors so that they flux abnormally high amounts of calcium into the cell during normal synaptic transmission. The mutant mice will be analyzed using a variety of techniques. The brains will be sectioned and examined for gross morphological changes and evidence of cell death. Behavioral tests including the Morris maze will be used to test for learning and memory function. After the mutant mice are characterized for neurodegenerative disease and cognitive deficits, attempts to cure the mutant mice will be carried out by expressing various potential ~protective~ or replacement proteins in the mouse brain using a number of different viral expression and cell graft systems.
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