GAD AND GABA IN ISLET CELL DEVELOPMENT AND FUNCTION

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Pancreatic beta cells and GABA-ergic neurons express the synthesizing enzyme for the inhibitory neurotransmitter GABA, glutamic acid decarboxylase. Pancreatic alpha cells and perhaps also delta cells express GABA/A receptors and GABA may function as a paracrine signaling molecule for communication among the endocrine cells in the pancreas. GAD is encoded by two distinct genes, GAD65 and GAD67, which differ mainly in the first 100 amino acids but share extensive homology in the remainder of the proteins. Human beta cells only express GAD65 and this form of the enzyme is a target of autoantibodies associated with early and late phases of beta cell destruction resulting in type 1 diabetes. Furthermore GAD65 has been shown to be an important T-cell autoantigen in the NOD-mouse model of type 1 diabetes. This project examines the role of GAD65 and GAD67, and GABA in islet cell development, function and beta cell autoantigenicity focusing on the cell biology of the GAD proteins and use of transgenic mouse models of aberrant GAD and GABA expression. The first aim is to characterize the topology of the GAD65 molecule which is synthesized as a hydrophilic soluble molecule, but is post- translationally modified and processed to become anchored to the membrane of synaptic like microvesicles. The second aim is to characterize the signaling and targeting sequences and the modification(s) that confer the subcellular localization of GAD65. The third aim is to characterize transgenic mouse models of over expression of GAD65 in beta cells and the consequences of such expression on islet cell development, function and autoantigenicity. The fourth aim is to generate transgenic mouse models over-expressing GAD65 late in development, and examine the consequences for tolerance development and autoantigenicity. The fifth aim is to characterize the islet cell and neurological function of GAD65 knock out mice and the consequences of rescue by wild type and mutant forms of GAD65. The overall goal is to elucidate the molecular mechanisms of function, subcellular localization and autoantigenicity of the GABA-ergic system in the pancreas.