Metabotropic Glutamate Receptors in Neurotoxicity

描述(申请人提供)：这项建议的目标是测试假设，即代谢性谷氨酸受体1是一种依赖受体，在没有谷氨酸的情况下诱导神经元凋亡，但在谷氨酸存在的情况下促进神经元的存活。MGluRt促凋亡作用的分子机制涉及其胞内C-末端结构域(CTD)被一种蛋白酶(Caspase)切割，然后该CTD与其他凋亡级联蛋白相互作用。MGluRI的毒性作用依赖于谷氨酸的存在、mGluRI的表达水平以及与激活PI3K-Akt通路的神经营养素受体的相互作用。MGluRI的表达水平由E2F1转录因子控制，受细胞周期相关机制的调控，在有丝分裂后神经元中，受各种毒性刺激的调控。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在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。其具体目的包括：(1)确定调节mGluRI表达的促凋亡和抗凋亡刺激。利用原代培养的神经细胞，我们将确定不同的凋亡刺激在促进mGluRI表达方面所起的作用。我们还将确定营养因子和其他抗凋亡药物在抑制mGluRI表达方面所起的作用。(2)探讨mGluRI表达的调控机制。利用分子技术和原代培养神经细胞，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和结构性质。利用原代培养的神经细胞和CHO细胞，我们将确定mGluRI的药理性质与其毒性作用和谷氨酸的保护作用有关。利用分子技术，我们还将确定作为依赖受体的mGluRI的结构特性。(4)探讨mGluRI的毒性机制。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，我们将鉴定mGluRI裂解位点和负责裂解mGluRI C-末端结构域的蛋白酶(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，这些蛋白可能与mGluRI的促凋亡作用有关。

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.