活化小胶质细胞转变ATP趋向性的分子机制研究

A hallmark of chronic CNS inflammation is the clustering of activated microglial cells at the disease sites,where they contribute to the pathogenesis of multiple neurodegenerative disorders including Alzheimer's and Parkinson diseases. ATP plays a pivotal role in triggering microglial migration towards the sites of diesease/injury. Ectonuleotidases,by hydrolyzing ATP to adenosine, are important factors that control the quantity of extracellular ATP. It is well-known that ATP-mediated P2 receptor signaling is key to the chemoattractive migration of microglia. Intriguingly,recent studies have shown that the pro-inflammatory liposaccride(LPS)-activated microglia showed repulsive migration in response to ATP, through the adenosine-P1 receptor signaling. Yet, the underlying regulatory mechanism remains unknown. We have found that prostate acid phosphatase (PAP), a novel type of ectonucleotidases, is robustly upregulated in LPS-activated microglia, suggesting that upregulated PAP may drive an enhanced adenosine-P1 receptor signaling, resulting in the chemotactic reversal of activated microglia. Based on these findings, this grant proposal will use real-time confocal and two-photon microscopy imaging, in combination with RNAi, knockout and pharmaceutical techniques, to further examine the role of PAP ectonucleotidase in the chemotactic switch of activated microglia, at cellular, organotypic and whole-animal levels. A migratory study of activated microglia from the perspective of ectonucleotidase not only provides new insights into the mechanisms underlying the migratory features of microglia, but also helps to elucidate the pathogenesis of CNS disorders in which the migration of activated microglia is involved.

活化小胶质细胞在病灶旁聚集是多种慢性CNS病变的特征，深入研究对阐明相关的病理机制意义重大。ATP是触发小胶质细胞迁移的关键分子，而外核苷酸酶水解ATP生成腺苷，是调控ATP浓度的重要因素。已知ATP介导的P2受体通路是诱导小胶质细胞向病损迁移的关键通路，新近研究却发现脂多糖活化的小胶质细胞能通过腺苷依赖的P1受体通路而逆转对ATP的趋向性，但调控机制未明。我们发现脂多糖活化小胶质细胞明显上调一种新型外核苷酸酶PAP，后者可能通过促进ATP水解生成腺苷，激活P1通路来改变细胞对ATP的趋向性。在此基础上，本项目将应用实时共聚焦和双光子显微成像、结合RNAi、基因敲除和药理阻断等技术，从细胞、活脑组织切片和整体动物三个方面系统探讨PAP对小胶质细胞迁移的调控。本项目以外核苷酸酶为切入点研究活化小胶质细胞迁移，将为小胶质细胞迁移的分子调控提供新思路，为阐明相关疾病的病理机制提供理论依据。

ATP是触发小胶质细胞迁移的关键分子，而外核苷酸酶水解ATP生成腺苷，是调控细胞外ATP和腺苷浓度的重要因素。已知帕金森病的动物模型及病人的中脑黑质纹状体通路存在广泛的腺苷A2A受体信号通路上调；黑质区域存在大量的小胶质细胞聚集，但其增强的腺苷来源一直及其与小胶质细胞迁移和聚集的不清楚。我们的研究显示在1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）诱导的帕金森病小鼠模型中，胞外-5'-核苷酸酶（CD73）产生的腺苷为腺苷A2A受体的激活提供了重要的来源，而上调的CD73和腺苷A2A受体则协同促进腺苷信号通路的增强。我们发现CD73来源的腺苷A2A受体信号通路可以调节小胶质细胞的免疫反应和形态动力学改变。CD73失活显著降低脂多糖（LPS）诱导小胶质细胞产生的促炎反应，但是在激光损伤及急性MPTP诱导的帕金森病小鼠模型中，CD73失活却增强小胶质细胞突起的延伸、运动和形态学转变。在帕金森病小鼠模型中，减少CD73来源的腺苷明显抑制了小胶质细胞调节的神经炎症，并提高了多巴胺神经元的存活及动物的运动能力。此外，CD73失活抑制了腺苷A2A受体的活化以及腺苷A2A受体介导的促炎反应，补充腺苷类似物却可以恢复这些效应，因此CD73引发了前馈自调节性的腺苷生成，从而活化腺苷A2A受体和促进神经炎症。我们首次发现，腺苷A2A受体的促炎效应是通过拮抗多巴胺调节的抗炎反应实现的，也就意味着腺苷和多巴胺信号通路二者间的平衡对小胶质细胞调节炎症反应十分重要。因此，我们的研究揭示了CD73调控的核苷酸代谢在调节神经炎症中的重要作用，并且以核苷酸代谢途径为靶点来限制腺苷产生和神经炎症，为帕金森病提供了一个有潜力的治疗策略。