Microglial activation by N-arachidonoyl glycine

描述（由申请人提供）：中枢神经系统的损伤或感染触发小胶质细胞定向迁移到死亡或死亡的神经元，在那里它们歧视性地吞噬并消除它们。这一过程受到严格控制，以维持对健康的相邻神经元的尽可能小的附带损害。在失去这种控制的情况下，小胶质细胞对死亡的神经元具有高度反应性，并引起继发性神经毒性，因此几乎与所有中枢神经系统神经病理过程（例如，阿尔茨海默病、帕金森病、肌萎缩侧索硬化症、艾滋病毒相关痴呆、克雅氏病）有关。这种极端的行为差异强调了理解特定信号系统的重要性，这些信号系统既可以招募小胶质细胞，也可以指导小胶质细胞选择性地适应其特征，以响应受损/死亡的神经元。我们最近证明了n -花生四烯醇酰甘氨酸（NAGly），一种在整个中枢神经系统产生的内源性脂质，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GPR18受体。以下建议将检验以下假设：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信号通路在表型上修饰小胶质细胞，使其在损伤部位发挥特殊作用；3)调节这些作用的神经元与小胶质细胞之间的通信是通过GPR18上的NAGly信号通路启动的。这将通过三个具体目标来实现。特异性目标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测试nagy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，该模型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，以确定这些脂质与小胶质细胞浸润相关的信号时间过程。特异性目标2:NAGly指定了什么小胶质细胞表型？NAGly刺激的BV-2小胶质细胞驱动迁移、MAPK激活和细胞增殖。在这里，我们将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），通过量化14种不同的细胞因子，确定NAGly（和相关脂质）是否同样启动促炎或抗炎表型。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测量前列腺素生产水平。特异性目标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敲除小鼠1)测量体内定向小胶质细胞迁移（WT与KO）， 2)生成CX3RC1+/GFP/GPR18 KO小鼠株，用于小胶质细胞驱动的中枢神经系统病理生理的未来研究。这些关于NAGly-GPR18受体信号传导的研究为理解神经元-小胶质细胞之间的通讯提供了新的途径，从而为抑制失调的小胶质细胞活性提供了潜在的治疗方法。

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.