NOVEL SCREENING ASSAY FOR EXCITATORY AMINO ACID ANTAGONISTS

EXCITATORY AMINO ACIDS (EAAS) ARE IMPLICATED IN THE PATHOLOGY OF A NUMBER OF HUMAN NEUROLOGICAL DISORDERS, INCLUDING EPILEPSY, ISCHEMIC BRAIN DAMAGE, AND NEURODEGENERATIVE DISEASES SUCH AS HUNTINGTON'S DISEASE AND ALZHEIMER'S DISEASE. THE ROLE OF EAAS IN THESE DISORDERS APPEARS TO INVOLVE ABNORMALLY ENHANCED ACTIVATION OF EAA RECEPTORS THAT LEADS TO EXCITOTOXIC CELL DAMAGE AND NEURONAL DEATH. RECENT EVIDENCE FROM ANIMAL MODELS INDICATES THAT SELECTIVE ANTAGONISTS FOR THESE RECEPTORS CAN BE EFFECTIVE IN THE PREVENTION OF RECEPTOR OVER-STIMULATION AND SUBSEQUENT CELL DEATH. THIS SUGGESTS THAT EAA ANTAGONISTS THAT PENETRATE THE BLOOD-BRAIN BARRIER AND ACT SELECTIVELY AT ONE OR MORE OF THE EAA RECEPTOR SUBTYPES MAY BE OF MAJOR CLINICAL IMPORTANCE FOR THE TREATMENT OF DISORDERS INVOLVING EAA NEUROTOXIC MECHANISMS. CURRENTLY, PRELIMINARY IDENTIFICATION OF EAA RECEPTOR ANTAGONISTS REQUIRES TIME-CONSUMING AND COMPLICATED ASSAYS USING LIVE ANIMALS OR ANIMAL TISSUES. WE PROPOSE TO DEVELOPA SIMPLIFIED ASSAY SYSTEM THAT IS SENSITIVE, RELIABLE, AND RAPID, AND WHICH DOES NOT REQUIRE CONTINUAL ANIMAL SACRIFICE. IN PHASE I OF THIS PROJECT WE WILL IDENTIFY AND CHARACTERIZE NEURONAL CELL LINES EXPRESSING FUNCTIONAL EAA RECEPTORS FROM A NOVEL GROUP OF NEURAL CELL LINES THAT HAVE NOT PREVIOUSLY BEEN CHARACTERIZED FOR THESE RECEPTORS. THESE LINES WERE ESTABLISHED BY THE INTRODUCTION OF ONCOGENES INTO PRIMARY BRAIN TISSUES USING RETROVIRAL VECTORS. IN PHASE II, WE WILL USE SCREENS EMPLOYING CELL LINES CONTAINING EAA RECEPTORS TO IDENTIFY NOVEL EAA ANTAGONISTS FROM NATURAL SOURCES (E.G., SPIDER VENOMS) AND SYNTHETIC COMPOUNDS UNDER ANALYSIS AT CNS RESEARCH.