Metabotropic Glutamate Receptors in Neurotoxicity

描述（由申请人提供）：本提案的目的是检验以下假设：代谢型谷氨酸受体1是一种依赖性受体，其在不存在谷氨酸的情况下诱导神经元凋亡，但在存在谷氨酸的情况下促进神经元存活。mGluRt促凋亡作用的分子机制涉及蛋白酶（半胱天冬酶）切割其细胞内C-末端结构域（CTD），然后该CTD与凋亡级联的其他蛋白相互作用。mGluRI的毒性作用取决于谷氨酸的存在、mGluRI表达水平以及与激活PI 3 K-Akt通路的神经营养因子受体的相互作用。mGluRI的表达水平由E2 F1转录因子控制，受细胞周期相关机制调节，在有丝分裂后神经元中受各种毒性刺激。在其作为依赖性受体的能力中，mGluRI可能在中枢神经系统的发育和成熟以及在由各种神经毒性因子损伤的神经元的凋亡消除中发挥关键作用。具体目的包括（1）鉴定调节mGluRI表达的促凋亡和抗凋亡刺激物。使用神经元细胞的原代培养物，我们将确定各种凋亡刺激在增强mGluRI表达中所起的作用。我们还将确定营养因子和其他抗凋亡药物在抑制mGluRI表达中所起的作用。(2)探讨mGluRI表达的调控机制。使用分子技术和神经元细胞的原代培养，我们将确定E2 F1转录因子调节mGluRI表达的分子机制，以及这种调节在介导各种毒性刺激的凋亡信号中的作用。(3)确定mGluRI作为依赖性受体的药理学和结构特性。使用神经元细胞和CHO细胞的原代培养物，我们将确定mGluRI的药理学特性，其毒性作用和谷氨酸的保护作用。使用分子技术，我们还将确定负责其作为依赖性受体的作用mGluRI的结构特性。(4)探讨mGluRI毒性作用的机制。使用CHO细胞中表达的突变受体以及培养的神经元细胞中的天然受体，我们将鉴定mGluRI切割位点和负责切割mGluRI C-末端结构域的蛋白酶（半胱天冬酶）的结构特性。使用双杂交系统，我们将确定与mGluRI CTD相互作用的推定蛋白质，这可能是负责mGluRI的促凋亡作用。

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.