Microglial activation by N-arachidonoyl glycine

描述（由申请人提供）：CNS中的损伤或感染触发小胶质细胞向死亡或垂死的神经元定向迁移，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，以便对健康的相邻神经元造成尽可能小的附带损害。在失去这种控制的情况下，小胶质细胞对垂死的神经元具有高度反应性，并引起继发性神经毒性，结果涉及几乎所有的CNS神经病理学过程（例如，阿尔茨海默病、帕金森病、肌萎缩侧索硬化、HIV相关痴呆、克雅氏病）。这种行为上的极端差异强调了理解特定信号系统的重要性，这些信号系统招募并指导小胶质细胞选择性地适应其特征以应对受损/死亡的神经元。我们最近证明，N-花生四烯酰甘氨酸（NAGly），一种在整个CNS中产生的内源性脂质，比任何已知的内源性脂质更有效地驱动BV-2小胶质细胞的迁移，可能是通过激活GPR 18受体。以下提案将检验以下假设：1）中枢神经系统损伤导致NAGly释放，从而驱动体内定向小胶质细胞迁移，2）NAGly信号传导表型修饰小胶质细胞，以在损伤部位发挥专门作用，以及3）调节这些作用的神经元与小胶质细胞通讯是通过GPR 18上的NAGly信号传导启动的。这将通过三个具体目标来实现。具体目标1：NAGly是否驱动CNS小胶质细胞的活化和迁移？为了通过免疫组织化学测试NAGly定向迁移的体内功效和特异性，我们将使用药物干预双侧颈总动脉闭塞（BCCAO）模型，其驱动视网膜中的小胶质细胞浸润。我们还将通过HPLC/MS/MS分析测量视网膜中NAGly和相关脂质的产生，以确定与小胶质细胞浸润相关的这些脂质的信号传导时间过程。具体目标2：NAGly指定什么样的小胶质细胞表型？用NAGly刺激的BV-2小胶质细胞驱动迁移、MAPK活化和细胞增殖。在此，使用原代小鼠小胶质细胞和小鼠细胞因子微阵列测定（US Biomax），我们将通过定量14种不同的细胞因子来确定NAGly（和相关脂质）是否同样引发促炎或抗炎表型。ICC将用于确定形态学的表型变化。ICC将用于确定形态学的表型变化。最后，还将通过HPLC/MS/MS测量前列腺素产生的水平。具体目标3：NAGly是否通过GPR 18诱导小胶质细胞迁移和启动CNS修复？我们将使用GPR 18敲除小鼠来1）测量体内定向小胶质细胞迁移（WT对KO）和2）产生CX 3RC 1 +/GFP/GPR 18 KO小鼠品系用于小胶质细胞驱动的CNS病理生理学的未来研究。NAGly-GPR 18受体信号传导的这些研究为理解神经元-小胶质细胞通信提供了一种新的途径，以开发旨在抑制失调的小胶质细胞活性的潜在治疗方法。

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.