脑卒中康复表达的生物活性脂类调节炎症基因表达和依赖PRG-1促进神经功能恢复的机理研究

This proposal employs lipidomics and imaging mass spectrometry technologies to determine changes of bioactive lipids following mouse transient cerebral ischemia. The aim is to identify significantly changed bioactive lipids during stroke recovery and to look for new lipid mediators associated with brain inflammation, neurodegeneration and regeneration. To support this application, pilot studies have been performed which identified over 500 lipid species in ischemic brains during the 1-28 days of reperfusion recovery period. Amongst these lipid species, 84 lipid species were found to have changed significantly during stroke recovery. However, whether these changes were associated with stroke outcomes remains unknown. ..In this proposal, we seek to continue to use the state-of-the-art lipidomics technology to profile changes of lipids and lipid rafts in the ischemic brain with a short-term goal to identify fingerprints/signatures lipid changes associated with neurodegeneration and neuroinflammation. The long-term goal is to explore these understandings to develop drugs targeting bioactive lipids to promote better brain recovery after stroke. ..We identified several groups of significantly changed lipids during reperfusion. Examples of these include PC(16:0/18:2), PC(16:0/16:0)，LPC(16:0)，PI(34:3/18:1), SM(d16:0/20:1), PE(16:0p/16:1) and MGDG(7:3/8:4). The role of these lipids as mediators for microglial activation, inflammation, axonal outgrowth, dendritic spine formation, growth cone chemotrophic guidance will be examined. Importantly, how bioactive lipids mediate hippocampal immediate early gene Arc expression to facilitate memory consolidation and recovery after stroke, in particular its molecular pathways, will be investigated. It is hypothesized that the plasticity-regulated-gene (PRG-1) plays a specific role at excitatory synapses on glutamatergic neurons where it interacts with bioactive phospholipids. Possible three downstream pathways will be examined, including, (1) modulating synaptic-localized calreticulin gene translation through its 5′UTR, (2) activating HDAC1/4 acetylation state for genetic changes affecting axonal outgrowth and neuritogenesis; (3) activating CAMK1 to induce F-actin changes thereby modulating the cytoskeleton of growth cone and synapses. In particular, experiments are designed to examine whether PRG-1 mediates the expression of hippocampal immediate early genes which are indicators for memory consolidation during stroke recovery. A battery of behavioral studies will be performed to confirm changes in cognitive functions during stroke recovery. ..Pilot studies from our laboratory established mass spectrometry-based imaging and lipidomics technologies. Brain lipids and lipid rafts will be isolated using customized methods from in vitro cultured cortical neurons and in vivo post-ischemic mouse brains. Their lipid composition will be determined using either a shotgun lipidomics approach and/or HPLC-ESI-MS/MS methods. In combination with molecular, cellular biology and behavioral approaches, we will determine lipoproteins or proteins associated with lipid rafts in order to tease out the molecular functions of this important membrane microdomain during the recovery response in stroke brain. ..To support this application, this laboratory has performed series of pilot studies and established all techniques required for the successful execution of the proposed experiments.

本项目拟利用小鼠大脑局灶缺血/再灌注模型，探讨脑卒中功能恢复中代谢脂组的变化，旨在发现表达的显著差异脂类并研究其作用机理。本课题组前期分析了小鼠卒中康复大脑1-28天中500种脂类的变化；将依此系统研究显著差异表达的生物活性脂诸如：PC(16:0/18:2), LPC(16:0)和MGDG(7:3/8:4)等是否通过cPLA2/NF-kB调节小胶质细胞炎症反应；是否通过plasticity-regulated gene-1（PRG-1）调节兴奋性神经元生长锥导向、突触形成、神经保护和网络再造；以及是否调控海马回突触内calreticulin以及与记忆巩固相关基因Arc的表达。分析膜脂筏内脂类分子与PRG-1、NRP-1/Fer激酶时空共表达图谱，验证膜脂筏作为功能康复信号转导中心假说。本研究的长期目标是发现生物活性脂类影响卒中功能康复新机理,为建立新的脑卒中康复医疗手段寻找新的突破口。

本项目利用小鼠大脑局灶缺血/再灌注模型，探讨脑卒中功能恢复中代谢脂组的变化，旨在发现表达的显著差异脂类、研究其作用机理。我们使用基于高灵敏度 UPLC-MS 的鸟枪脂质组学策略，半定量地测量了从短暂的大脑中动脉闭塞 (MCAO) 中恢复的小鼠大脑皮层中的差异脂质含量，分析了小鼠卒中康复大脑1-28天中近500种脂类的变化，并且系统的研究了一批显著差异表达的生物活性脂诸如。 比如， PC(16:0/18:2), LPC(16:0)和MGDG(7:3/8:4)等。使用涉及主成分分析 (PCA) 和正交偏最小二乘判别分析 (OPLS-DA) 的类别类比软独立建模 (SIMCA) 方法查询脂质组学数据。对 578 种已确认脂质的统计显示，84 种脂质在 MCAO/再灌注过程中发生了差异变化。脂质最大幅度动态变化发生在 MCAO 后 1 到 7 天之间，而浓度在 MCAO 后 14 天和 28 天时下降到假手术组水平。定量分析显示磷脂酰胆碱 (PC)(16:0/16:0) 的减少与溶血磷脂酰胆碱 (LPC)(16:0) 水平的增加在 1 到 7 之间存在强烈的正相关性。抑制 cPLA2 阻止了 PC(16:0/16:0) 和 LPC(16:0) 之间比率的变化，表明兰德（Lands）的 PC 循环发生了变化。一系列体外研究表明，LPC(16:0) 而不是 PC(16:0/16:0) 会通过引起细胞内钙流入而损害神经元生长锥的完整性和神经元活力。相反，PC(16:0/16:0) 显着抑制小胶质细胞分泌 IL-1β 和 TNF-α，从而限制神经炎症通路。总之，这些数据支持由 Lands 循环维持的 PC(16:0/16:0) 和 LPC(16:0) 之间比例失衡在缺血恢复期间神经元损伤和小胶质细胞介导的炎症反应中的作用。本研究的长期目标是发现生物活性脂类影响卒中功能康复新机理,为建立新的脑卒中康复医疗手段寻找新的突破口。本课题组完成了所有本项目计划任务目标。