Metabotropic Glutamate Receptors in Neurotoxicity

描述（由申请人提供）：本提案的目的是验证代谢性谷氨酸受体1是一种依赖性受体的假设，该受体在谷氨酸缺失时诱导神经元凋亡，但在谷氨酸存在时促进神经元存活。mGluRt促凋亡作用的分子机制涉及其细胞内c -末端结构域（CTD）被蛋白酶（caspase）切割，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的表达水平以及与激活PI3K-Akt通路的神经营养因子受体的相互作用。mGluRI的表达水平受E2F1转录因子控制，受细胞周期相关机制调节，并且在有丝分裂后神经元中受各种毒性刺激的调节。作为一种依赖受体，mGluRI可能在中枢神经系统的发育和成熟以及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。利用神经细胞的原代培养，我们将确定各种凋亡刺激在增强mGluRI表达中的作用。我们还将确定营养因子和其他抗凋亡药物在抑制mGluRI表达中的作用。(2)确定mGluRI表达的调控机制。利用分子技术和神经细胞原代培养，我们将确定E2F1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作为依赖受体的药理学和结构特性。利用神经细胞和CHO细胞的原代培养，我们将确定mGluRI与其毒性作用和谷氨酸保护作用相关的药理学特性。利用分子技术，我们还将确定mGluRI作为依赖受体的结构特性。(4)确定mGluRI毒性作用机制。利用CHO细胞中表达的突变受体以及培养的神经细胞中的天然受体，我们将确定mGluRI切割位点和负责mGluRI c端区域切割的蛋白酶（caspase）的结构特性。利用双杂交系统，我们将鉴定可能与mGluRI CTD相互作用的蛋白，这些蛋白可能负责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.