Functional Effects of Adenosine A2A Receptor Activation on Microglia

描述(申请人提供)：小胶质细胞，中枢神经系统的常驻免疫细胞，通常存在于“静止”状态，胞体较小，突起分叉。在损伤的情况下，如小血管破裂，小胶质细胞将它们的突起延伸到损伤部位，并似乎通过吞噬来清除细胞碎片。这种定向过程的延伸是由受损细胞释放的ATP和激活小胶质细胞上的P2Y12受体介导的。然而，在长时间的损伤和许多神经退行性疾病中，小胶质细胞呈现阿米巴样的“激活”表型，其特征是细胞体较大，突起迟缓，并释放促炎细胞因子。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导致过程回缩和迁移。这种反应的转换被认为是小胶质细胞激活时同时发生的P2Y12受体下调和A2a受体上调的结果。在此，我们假设A2a受体的激活通过在体外增加细胞因子和活性氧物种(ROS)的分泌以及改变体内小胶质细胞对损伤的反应能力来促进激活的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受体激活是否改变了激活的小胶质细胞分泌细胞因子和/或ROS的模式；以及(2)静息和激活的小胶质细胞对神经元损伤的反应是否不同。为了确定A2a受体在调节重要的小胶质细胞功能中可能发挥的作用，我们将研究A2a受体激活对有代表性的细胞因子和ROS产生的影响。然后，我们将在体外检测腺苷梯度对小胶质细胞运动的影响。最后，我们将在体内对表达GFP的小胶质细胞进行双光子成像，以确定小胶质细胞上A2A受体的激活是否调节了它们对神经元损伤的反应。这些实验的成功完成将阐明A2A受体在激活的小胶质细胞中的功能，并提供更好的理解，以治疗为目的靶向这些受体可能如何影响中枢神经系统的小胶质细胞。 与公共健康相关：这项拟议研究的目标是研究大脑自身免疫系统的调节机制。这项研究的结果可能会对免疫系统在神经退行性疾病发展中的作用产生影响，并提供一种调节疾病进展的方法。此外，这项研究将为咖啡因等常见环境因素如何影响大脑的免疫细胞，从而影响大脑病理提供新的见解。

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. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to study a mechanism of regulation of the brain's own immune system. The results from the research might have implications on the role of the immune system in the development of neurodegenerative diseases, and provide a way modulate disease progression. In addition, the research will provide new insights on how common environmental agents such as caffeine might affect the immune cells of the brain, and thus brain pathologies.