Metabotropic Glutamate Receptors in Neurotoxicity

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DESCRIPTION (provided by applicant): The goal of this proposal is to test the hypothesis that the metabotropic glutamate receptor 1 is a dependence receptor which induces neuronal apoptosis in the absence of glutamate, but promotes neuronal survival in the presence of glutamate. The molecular mechanism of the pro-apoptotic action of mGluRt involves the cleavage of its intracellular C-terminal domain (CTD) by a protease (caspase) followed by the interaction of this CTD with other proteins of the apoptotic cascade. The toxic action of mGluRI depends on the presence of glutamate, the levels of mGluRI expression, and on interactions with neurotrophin receptors activating the PI3K-Akt pathway. The levels of mGluRI expression are controlled by the E2F1 transcription factor, regulated by cell cycle-associated mechanisms, and, in postmitotic neurons, by various toxic stimuli. In its capacity as a dependence receptor mGluRI may play a pivotal role in the development and maturation of the central nervous system and in the apoptotic elimination of neurons damaged by various neurotoxic factors. The specific aims include (1) To identify the pro- and anti-apoptotic stimuli regulating mGluRI expression. Using primary cultures of neuronal cells we will identify the role played by various apoptotic stimuli in enhancing mGluRI expression. We will also determine the role played by trophic factors and other anti-apoptotic agents in suppressing mGluRI expression. (2) To determine the mechanisms regulating mGluRI expression. Using molecular techniques and primary cultures of neuronal cells we will determine the molecular mechanisms whereby the E2F1 transcription factor regulates mGluRI expression and the role of this regulation in mediating the apoptotic signal of various toxic stimuli. (3) To determine the pharmacological and structural properties of mGluRI as a dependence receptor. Using primary cultures of neuronal cells and CHO cells we will determine the pharmacological properties of mGluRI related to its toxic action and to the protective effect of glutamate. Using molecular techniques we will also determine the structural properties of mGluRI responsible for its action as a dependence receptor. (4) To determine the mechanism of mGluRI toxicity. Using mutated receptors expressed in CHO cells, as well as, native receptors in cultured neuronal cells we will identify the structural properties of the mGluRI cleavage site and the protease (caspase) responsible for the cleavage of mGluRI C-terminal domain. Using the two-hybrid system we will identify the putative proteins interacting with the mGluRI CTD which may be responsible for the pro-apoptotic action of mGluRI.