During the current grant period, we established that GABA synthesis depends on two forms of glutamate decarboxylase (GAD65 and GAD67), which differ in size, sequence, intracellular location, and in their interactions with the obligate cofactor, pyridoxal phosphate. Using a retroviral vector, we have engineered a number of cell lines to produce either or both GADs and shown that these cells produce GABA both in culture and after transplantation into lesioned rats. The proposed studies will extend this work to problems specifically related to epilepsy. The rationale of the proposed approach is the finding from several other laboratories that increased GABA levels in the substantia nigra suppresses chemically and electrically induced seizures as well as the development of amygdaloid kindling.
The Specific Aims for the next grant period are: 1. to use GADs produced in engineered bacteria and eukaryotic cells to study the regulation of GAD enzymatic activity both in vitro and in cells; 2. to use cell lines engineered to produce GAD to understand (a) differences between the two GADs in intracellular targeting and (b) the contributions of each GAD to vesicular and non-vesicular GABA release; 3. to transplant into the substantia nigra cells engineered to produce GABA (either constitutively or in an inducible manner) in order to study the effect of in situ GABA release on the propagation of seizures evoked by electroshock; and 4. to study the effects of grafted GABA-producing cells on the expression of GABA- and glutamate-related genes and on the development of amygdaloid kindling. The proposed experiments will test the following hypotheses: (1) that the cellular regulation of GAD activity involves changes in the relative rates of decarboxylation and transamination of the GAD-PLP-glutamate complex, with transamination leading to the formation of inactive apoGAD; (2) that the two GADs are responsible for the production of distinct GABA pools, with GAD67 contributing primarily to the production of non-vesicular GABA and GAD65 to the production of vesicular GABA; (3) that GABA produced by transplanted cells can alter gene expression of GABA- and glutamate-related genes and can suppress seizure propagation; and (4) that changes in the expression of genes and proteins associated with the production and action of GABA and glutamate accompany the development of kindled seizures.
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