Regulation of Neuroprotective Lipid Sgnals by Spatially Distinct NMDA Receptors

描述（由申请人提供）： 这项研究是长期努力的一部分，旨在了解脂质信号传导机制如何决定受兴奋性细胞死亡疾病影响的脑细胞的命运。突触NMDA受体的刺激促进神经元存活，而突触外NMDA受体的刺激导致显性的促死亡信号传导。然而，决定这两种结果的分子途径尚未完全了解。两个很大程度上不重叠的基因表达程序介导的细胞存活和细胞死亡，分别由这些空间上不同的NMDA受体亚群的信号转导。神经保护D1（NPD 1）是一种立体特异性脂质介质，来源于二十二碳六烯酸（DMA），激活人脑和视网膜细胞中的促生存基因表达程序。NPD1介导来自突触NMDA受体的促存活信号吗？将使用原代海马细胞培养物研究突触和突触外NMDA受体对NPD 1生物利用度的控制。实验将集中于脑源性神经营养因子（BDNF）对NPD 1池大小的调节，BDNF是来自突触NMDA受体的促存活信号传导的介体，并且是NPD 1合成的已知激动剂。实验将采用细胞和分子生物学和生物化学技术，基于HPLC-ESI串联质谱的脂质组学分析，以及使用15-脂氧合酶-1（15-LOX-1）缺陷小鼠，这些小鼠不能合成NPD 1。所提出的研究的结果将证明，促生存信号从突触NMDA受体介导的NPD 1池的大小通过增加BDNF的表达增强。此外，15-LOX-1缺陷型神经元培养物将显示出比野生型培养物显著更易受兴奋性毒性损伤，并且NPD 1将显示出在体外和体内减弱兴奋性毒性损伤。

DESCRIPTION (provided by applicant): The proposed research is part of a long-term effort to understand how lipid signaling mechanisms determine the fate of brain cells affected by diseases which exhibit excitotoxic cell death. Stimulation of synaptic NMDA receptors promotes neuronal survival, while stimulation of extrasynaptic NMDA receptors leads to dominant, pro-death signaling. However, the molecular pathways that determine these two outcomes are not fully understood. Two largely non-overlapping gene expression programs mediating cell survival and cell death, respectively, are induced by signaling from these spatially distinct NMDA receptor subpopulations. Neuroprotection D1 (NPD1) is a stereospecific lipid mediator derived from docosahexaenoic acid (DMA) that activates a pro-survival gene expression program in human brain and retinal cells. Does NPD1 mediate pro- survival signals from synaptic NMDA receptors? Control of NPD1 bioavailability by synaptic and extrasynaptic NMDA receptors will be investigated using primary hippocampal cell cultures. Experiments will focus on NPD1 pool size regulation by brain-derived neurotrophic factor (BDNF), which is a mediator of pro- survival signaling from synaptic NMDA receptors and a known agonist of NPD1 synthesis. Experiments will employ techniques from cell and molecular biology and biochemistry, HPLC-ESI tandem mass spectrometry-based lipidomic analysis, and the use of 15-lipoxygenase-1 (15-LOX-1) deficient mice, which are incapable of NPD1 synthesis. The results of the proposed studies will demonstrate that pro-survival signaling from the synaptic NMDA receptor is mediated through enhancement of the NPD1 pool size via increased expression of BDNF. Additionally, 15-LOX-1 deficient neuronal cultures will be shown to be significantly more vulnerable to excitotoxic injury than wild type cultures, and NPD1 will be shown to attenuate excitotoxic injury in vitro and in vivo.