Functional Effects of Adenosine A2A Receptor Activation on Microglia

描述（由申请人提供）：小胶质细胞是中枢神经系统的常驻免疫细胞，通常以小细胞体和分支过程的“休息”状态存在。在损伤的情况下，如小血管破裂，小胶质细胞将其过程扩展到损伤部位，并通过吞噬作用清除细胞碎片。这种定向过程扩展是由受损细胞释放ATP和小胶质细胞上P2Y12受体的激活介导的。然而，在长期损伤和许多神经退行性疾病中，小胶质细胞呈现变形虫“活化”表型，其特征是细胞体变大，发育迟缓，释放促炎细胞因子。令人惊讶的是，在体外激活的小胶质细胞的脂多糖（LPS）模型中，ATP引起过程收缩和远离其来源的迁移。这种反应的转换被认为是P2Y12受体下调和A2A受体同时上调的结果，这些上调发生在小胶质细胞激活过程中。在这里，我们假设A2A受体激活通过增加细胞因子和活性氧（ROS）的分泌，以及改变小胶质细胞对体内损伤的反应能力，有助于激活小胶质细胞的促炎表型。为了验证这一假设，我们建议检验(1)A2A受体激活是否会改变激活的小胶质细胞的细胞因子和/或ROS分泌模式；(2)静息和活化的小胶质细胞对神经元损伤的反应是否不同。我们将研究A2A受体激活对代表性细胞因子和ROS产生的影响，以确定A2A受体在调节重要的小胶质细胞功能方面可能发挥的作用。然后，我们将研究腺苷梯度对体外小胶质细胞运动的影响。最后，我们将对体内表达gfp的小胶质细胞进行双光子成像，以确定小胶质细胞上A2A受体的激活是否会调节它们对神经元损伤的反应。本实验的成功完成将阐明A2A受体在活化的小胶质细胞中的功能，并更好地了解靶向这些受体的治疗目的如何影响中枢神经系统中的小胶质细胞。

DESCRIPTION (provided by applicant): Microglia, the resident immune cells of the central nervous system, normally exist in a "resting" state with small cell bodies and ramified processes. In cases of injury, such as rupture of small blood vessels, microglia extend their processes to the site of injury and appear to clear cellular debris by phagocytosis. This directional process extension is mediated by ATP released from damaged cells and activation of P2Y12 receptors on microglia. However, in prolonged injury and in many neurodegenerative conditions microglia assume an amoeboid "activated" phenotype characterized by larger cell bodies, stunted processes, and release of pro-inflammatory cytokines. Surprisingly, ATP causes process retraction and migration away from its source in the lipopolysaccharide (LPS) model of activated microglia in vitro. This switch in response is thought to be the consequence of P2Y12 receptor downregulation and concurrent A2A receptor upregulation that occur with microglial activation. Here, we hypothesize that A2A receptor activation contributes to the pro-inflammatory phenotype of activated microglia by increasing cytokine and reactive oxygen species (ROS) secretion in vitro and altering the ability of microglia to respond to injury in vivo. In order to test this hypothesis, we propose to examine (1) if A2A receptor activation changes the pattern of cytokine and/or ROS secretion by activated microglia; and (2) whether resting and activated microglia respond differently to neuronal injury. The effect of A2A receptor activation on the production of representative cytokines and ROS will be examined in order to determine the role that A2A receptors might play in modifying important microglial functions. Then, we will examine the effects of an adenosine gradient on microglial motility in vitro. Finally, we will perform two photon imaging of GFP-expressing microglia in vivo to determine if activation of A2A receptors on microglia modulates their response to neuronal damage. The successful completion of the proposed experiments will elucidate the function of A2A receptors in activated microglia and provide a better understanding of how targeting these receptors for therapeutic purposes might affect microglia in the CNS.