N-acetylaspartylglutamate (NAA) is the most prevalent and widely distributed peptide neurotransmitter in the mammalian nervous system. The PI's laboratory has demonstrated that this peptide is concentrated in neurons and localized in synaptic vesicles, is released in a calcium-dependent manner from axon endings following initiation of action potentials, is equipotent to glutamate as an agonist at the metabotropic glutamate receptor type 3 (mGluR3), and is inactivated by hydrolysis via membrane-bound extracellular peptidase activity (NAAGp). The peptidase is a site for modulating the synaptic concentration of NAAG, thus providing a significant strategy for defining the role of this peptide at identified synapses. This approach permits testing of the hypothesis that a primary action of NAAG is inhibition of synaptic release by activation of mGluR3 receptors. Given the codistribution of NAAG with a spectrum of amine transmitters in neurons, demonstration of a role in regulation of synaptic release will provide a foundation for development of new pharmacological strategies to regulate nervous system function. The PI's laboratory cloned a cDNA from rat hippocampus that codes for a NAAG peptidase activity similar to that identified in mammalian brain. This enzyme, termed NAAGpI, and a related peptidase, NAAGpII, which has been identified in brain by RT-PCR, will be characterized along with related enzymes that may be identified in EST databases. The cellular localization of these NAAGps will be determined in cell cultures and in individual cells by single cell RT-PCR in order to test the hypothesis that they are expressed exclusively by glia. Two compounds have been synthesized by a collaborator, Alan Kozikowski, at Georgetown. They completely inhibit rat brain NAAGp activity, providing the opportunity to explore the physiological consequences of NAAGp inhibition in cell cultures and slice preparations of rat brain. Additionally, we will test the hypothesis that NAAGpI has an exclusive or significant role in the inactivation of synaptic NAAG in a mouse line that is null mutant for this peptidase. Heterozygous mice have been produced by a collaborator, Warren Heston, to study cancer cell biology and the PI's laboratory will characterize the nervous system consequences of this gene loss. The efficacy of NAAG in suppression of transmitter release and long-term potentiation (LTP) will be defined further in rat brain slices and in cell cultures. The peptidase inhibitors, FN6 and FN11, will be used to test the hypothesis that endogenous NAAG participates in the regulation of LTP and transmitter release.
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