A Genetic Model of NMDA Receptor Function in Cell Death

描述(申请人提供)：兴奋性细胞死亡是阿尔茨海默氏症或帕金森氏症等神经退行性疾病以及神经系统受到侮辱后(如中风或酗酒)导致神经组织丧失的原因。这种类型的细胞死亡是由谷氨酸受体，特别是N-甲基-D-天冬氨酸受体(NMDAR)过度刺激钙摄取引起的。尽管兴奋性细胞死亡在医学上具有重要意义，但受体激活下游事件的连贯模型一直迟迟没有出现。由于病理条件下细胞死亡的复杂性，以及缺乏允许对这一过程进行遗传解剖的可获得的动物模型，进展受到了阻碍。这个项目是基于一项发现，即在遗传模式生物黑腹果蝇中，NMDAR是非病理性程序性细胞死亡所必需的。幼虫唾液腺在变态过程中的死亡是细胞死亡研究中的一个重要模型系统，但当NMDAR活性降低时，这种死亡就会失败。这为利用模式生物研究的强大工具和方法研究NMDAR在细胞死亡中的作用提供了可能。作为这项研究的第一个结果，蛋白质酪氨酸磷酸酶Ptpmeg被确定为NMDAR在细胞死亡控制中的假定合作伙伴。缺乏Ptpmeg的唾液腺不会死亡，并且携带的钙负荷较低。Ptpmeg控制细胞死亡是一项新的功能，可能延伸到其人类同源物，这些同源物在癌症中调节失调，被怀疑是肿瘤抑制因子。该项目的广泛目标是确定NMDAR和Ptpmeg如何在细胞死亡控制中合作，以及这两种蛋白质如何影响进化保守的细胞死亡计划中的步骤。具体地说，该项目将(1)在转基因动物中使用遗传方法来确定NMDAR和Ptpmeg如何在调节细胞内钙离子和细胞死亡方面进行合作。联合免疫定位和下拉试验将用于检查NMDAR和Ptpmeg之间的物理相互作用。用磷酸特异性抗体染色将揭示NMDAR酪氨酸磷酸化形式的表达谱及其对Ptpmeg的依赖。(2)将在使用钙缓冲蛋白的转基因动物中研究钙在细胞死亡中的作用。NMDAR信号的下游靶点将通过分析NMDAR活性降低对钙依赖的细胞清除途径以及凋亡和自噬标记物的影响来确定。(3)生化和遗传分析将被用来确定受缺乏Ptpmeg影响的凋亡和钙信号通路中的步骤。总之，拟议的研究结果可能会揭示NMDAR和Ptpmeg在进化上保守的细胞死亡控制的基本机制。因此，该项目将有助于了解这些蛋白质在病理条件下的功能，并有助于开发治疗措施。

DESCRIPTION (provided by applicant): Excitatory cell death is responsible for the loss of nervous tissue in neurodegenerative disorders such as Alzheimer's or Parkinson's disease and after insults to the nervous system, for instance through stroke or alcohol abuse. This type of cell death is caused by an overstimulation of calcium uptake through glutamate receptors, in particular the N-methyl-D-aspartate receptor (NMDAR). Despite the medical importance of excitatory cell death, a coherent model of the events downstream of receptor activation has been slow to emerge. Progress has been hampered by the complexity of cell death under pathological conditions and the lack of an accessible animal model that allows a genetic dissection of the process. This project is based on the discovery that NMDAR is required for non-pathological programmed cell death in the genetic model organism Drosophila melanogaster. Death of the larval salivary glands during metamorphosis, which constitutes an important model system in cell death research, fails when NMDAR activity is reduced. This opens up the possibility to study the role of NMDAR in cell death using the powerful tools and approaches of model organism research. As a first result of this research, the protein tyrosine phosphatase Ptpmeg was identified as a putative partner of NMDAR in cell death control. Salivary glands deficient in Ptpmeg do not die and carry a lowered calcium load. Cell death control by Ptpmeg is a novel function that likely extends to its human homologs, which are dysregulated in cancers and suspected to be tumor suppressors. The broad objective of this project is to determine how NMDAR and Ptpmeg cooperate in cell death control and how the two proteins affect steps in the evolutionarily conserved cell death program. Specifically, the project will (1) use genetic approaches in transgenic animals to determine how NMDAR and Ptpmeg cooperate in regulating intracellular Ca2+ and cell death. Co- immunolocalization and pull-down assays will be used to examine physical interactions between NMDAR and Ptpmeg. Staining with a phospho-specific antibody will reveal the expression profile of the tyrosine-phosphorylated form of NMDAR and its dependence on Ptpmeg. (2) The role of Ca2+ in cell death will be examined in transgenic animals using a Ca2+-buffer protein. Downstream targets of NMDAR signaling will be identified by analyzing the effects of reduced NMDAR activity on Ca2+-dependent cell clearance pathways and markers of apoptosis and autophagy. (3) Biochemical and genetic assays will be used to identify steps in apoptotic and Ca2+-signaling pathways that are affected by a lack of Ptpmeg. Together, the results of the proposed research are likely to bring to light evolutionarily conserved, basic mechanisms of cell death control by NMDAR and Ptpmeg. Thus, the project will help understand the function of these proteins under pathological conditions and aid in the development of therapeutic measures.