Microglial activation by N-arachidonoyl glycine

描述(申请人提供)：中枢神经系统的损伤或感染触发小胶质细胞进行定向迁移，向死亡或濒临死亡的神经元迁移，在那里它们有区别地吞噬和消除它们。这一过程受到严格控制，以便将对健康的相邻神经元的附带损害降至最低。在失去这种控制的地方，小胶质细胞对死亡的神经元高度反应，引发继发性神经毒性，结果涉及几乎所有的中枢神经病理过程(例如，阿尔茨海默病、帕金森病、肌萎缩侧索硬化症、艾滋病毒相关性痴呆、克雅病)。这种极端的行为差异强调了了解特定的信号系统的重要性，这些信号系统既招募又指示小胶质细胞选择性地调整其特征，以应对受损/死亡的神经元。我们最近证实，N-花生四烯酸甘氨酸(NAGLY)是一种遍及中枢神经系统的内源性脂质，可能通过激活GPR18受体比任何已知的内源性脂质更有效地推动BV-2小胶质细胞的迁移。下面的提案将检验以下假设：1)中枢神经系统的损伤导致nAGLY的释放，从而驱动体内定向的小胶质细胞迁移；2)nAGLY信号典型地修饰小胶质细胞，使其在损伤部位发挥特殊作用；以及3)调节这些活动的神经元到小胶质细胞的通讯是通过GPR18处的nAGLY信号启动的。这将通过三个具体目标来实现。特定目的1：nAGLY是否驱动中枢神经系统小胶质细胞的激活和迁移？为了通过免疫组织化学方法测试nAGLY定向迁移的体内有效性和特异性，我们将对双侧颈总动脉阻塞(BCCAO)模型进行药物干预，以驱动视网膜的小胶质细胞渗透。我们还将通过HPLC/MS/MS分析来测量视网膜中nAGLY和相关脂类的产生，以确定这些脂类与小胶质细胞渗透相关的信号时程。具体目标2：nAGLY指定的小胶质细胞表型是什么？NAGLY刺激的BV-2小胶质细胞促进迁移、MAPK激活和细胞增殖。在这里，使用原代小鼠小胶质细胞和小鼠细胞因子微阵列分析(US Biomax)，我们将通过量化14种不同的细胞因子来确定nAGLY(和相关脂类)是否同样启动促炎或抗炎表型。ICC将被用来确定形态的表型变化。ICC将被用来确定形态的表型变化。最后，还将通过HPLC/MS/MS测量前列腺素的产生水平。具体目的3：nAGLY是否通过GPR18诱导小胶质细胞迁移和启动中枢神经系统修复？我们将使用GPR18基因敲除小鼠来1)测量体内小胶质细胞的定向迁移(WT和KO)，以及2)产生一个品系的CX3RC1/GFP/GPR18 KO小鼠，用于未来小胶质细胞驱动的中枢神经系统病理生理的研究。这些对nAGLY-GPR18受体信号转导的研究为了解神经元-小胶质细胞之间的联系提供了一条新的途径，为开发旨在抑制小胶质细胞活性失调的潜在疗法提供了新的途径。 与公共健康相关：大脑和脊髓拥有特殊的免疫细胞，称为小胶质细胞，对损伤和感染做出反应。受损的神经元通过释放化学分子与小胶质细胞通信，这些化学分子发出信号让它们采取行动；然而，我们对这些信号的性质以及我们如何最好地从医学上影响它们知之甚少。这一提议概述了这种神经元到小胶质细胞的一种以前未知的通信形式(即一种新的信号分子：N-花生四烯酸甘氨酸及其细胞受体GPR18，它触发小胶质细胞向信号源迁移以开始修复。

DESCRIPTION (provided by applicant): Damage or infection in the CNS triggers microglia to undergo directed migration towards dead or dying neurons, where they discriminately engulf and eliminate them. This process is tightly controlled in order to sustain the least possible collateral damage to healthy, adjacent neurons. Where there is a loss of this control, microglia are highly reactive to dying neurons and provoke secondary neurotoxicity and as a result are implicated in nearly all CNS neuropathological processes (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, HIV-related dementia, Creutzfeld-Jakob disease). This extreme difference in behavior underscores the importance of understanding the specific signaling systems that both recruit and instruct microglia to selectively adapt their characteristics in response to damaged/dying neurons. We recently demonstrated that N-arachidonoyl glycine (NAGly), an endogenous lipid produced throughout the CNS, drives migration of BV-2 microglial cells more potently than any known endogenous lipid likely through activation of GPR18 receptors. The following proposal will examine the hypotheses that 1) damage to the CNS causes release of NAGly, which drives directed microglial migration in vivo, 2) NAGly signaling phenotypically modifies microglia to perform specialized roles at sites of damage, and 3) the neuronal to microglial communication regulating these actions is initiated through NAGly signaling at GPR18. This will be achieved through three specific aims. Specific Aim 1: Does NAGly drive the activation and migration of CNS microglia? To test the in vivo efficacy and specificity of NAGly-directed migration by immunohistochemistry, we will use pharmacological interventions with a bilateral common carotid artery occlusion (BCCAO) model that drives microglial infiltration in the retina. We will also measure production of NAGly and related lipids in the retina by HPLC/MS/MS analysis to determine the signaling time course of these lipids associated with microglial infiltration. Specific Aim 2: What microglia phenotype does NAGly specify? BV-2 microglia cells stimulated with NAGly drives migration, MAPK activation, and cell proliferation. Here, using primary mouse microglia and mouse cytokine microarray assays (US Biomax) we will determine if NAGly (and related lipids), likewise, initiate pro- or anti- inflammatory phenotypes by quantifying 14 different cytokines. ICC will be used to determine phenotypic changes in morphology. ICC will be used to determine phenotypic changes in morphology. Finally, levels of prostaglandin production will also be measured via HPLC/MS/MS. Specific Aim 3: Does NAGly induce microglial migration and initiation of CNS repair through GPR18? We will use GPR18 knockout mice to 1) measure directed microglial migration in vivo (WT verses KO) and 2) generate a strain of CX3RC1+/GFP/GPR18 KO mouse for future studies of microglia-driven CNS pathophysiologies. These studies of NAGly-GPR18 receptor signaling supply a novel route for understanding neuronal-microglial communication to exploit for potential therapeutics aimed at suppressing dysregulated microglial activity. PUBLIC HEALTH RELEVANCE: The brain and spinal cord possess specialized immune cells, named microglia, that respond to damage and infection. Damaged neurons communicate to microglia by releasing chemical molecules that signal for them to act; however, we have a poor understanding of the nature of these signals and how we can best influence them medicinally. This proposal outlines a previously unknown form of this neuron to microglia communication (i.e., a novel signaling molecule: N-arachidonoyl glycine, and its cellular receptor, GPR18, that triggers microglia to migrate towards the source of the signal in order to begin repair.