E-Prostanoid Receptor Subtype 2 (EP2) Regulation of Microglial Activation

描述(由申请人提供)：激活小胶质细胞中的先天免疫反应与几种神经退行性疾病的启动或进展有关。也许在阿尔茨海默病(AD)模型中最好的研究是先天免疫激活，已被证明对神经元既有好处也有坏处。有益的作用与Abeta多肽的神经毒性物质的清除有关，而旁分泌对神经元的损害是由于包括活性氧和氮物种在内的各种有毒物质的阐述造成的。许多学术和药物实验室的一个主要目标是确定在抑制旁分泌神经毒性的同时增强或维持天然免疫激活的有益作用的方法。最近，我们证明了遗传消融小鼠原代小胶质细胞的EP2受体导致了高度理想的双重表型，即增加了Abeta物种的吞噬能力，并完全阻断了旁分泌神经毒性。其他人用不同的方法验证了我们的发现。综上所述，这些数据有力地支持EP2受体作为一个非常有希望的靶点来操纵AD和其他神经退行性疾病的小胶质细胞先天免疫反应。EP2介导的信号与Abeta吞噬和旁分泌神经毒性相关的机制尚不清楚。我们对小鼠小胶质细胞原代表达的初步数据确定了一个与EP2密切相关的候选基因。据我们所知，目前还没有关于该基因在包括小胶质细胞在内的任何脑细胞中作用的报道。然而，根据新出现的数据，我们假设它的表达和活性与EP2信号机制有关，它可能是消融EP2产生高度理想的双重表型的关键因素，即增强Aβ吞噬功能和降低旁分泌神经毒性。我们将通过以下具体目标来验证这一假设：1)在Abeta治疗前后，对我们的候选mRNA和蛋白在野生型和EP2-/-原代小鼠小胶质细胞中的表达进行体外验证。2)对野生型和EP2-/-小鼠骨髓移植前后野生型和转基因AD小鼠模型的脑内局部和细胞分布进行定位。3)利用shRNA敲除，确定其在EP2-/-双重表型背景下在敲除小鼠和小胶质细胞系中表达的功能性。

DESCRIPTION (provided by applicant): Activation of innate immune response in microglia is associated with the initiation or progression of several neurodegenerative diseases. Perhaps best studied in models of Alzheimer's disease (AD), innate immune activation has been shown to have both beneficial and deleterious effects on neurons. Beneficial effects are related to the clearance of nerurotoxic species of Abeta peptides, while paracrine damage to neurons results from the elaboration of a variety of toxic substances including reactive oxygen and nitrogen species. A major goal of many academic and pharmaceutical laboratories is to identify means to augment or maintain the beneficial actions of innate immune activation while suppressing paracrine neurotoxicity. Recently we demonstrated that genetic ablation of the EP2 receptor from primary mouse microglia resulted in the highly desirable dual phenotype of an increase in phagocytosis of Abeta species and complete blockade of paracrine neurotoxicity. Others have validated our findings using different methods. Taken together, these data strongly support EP2 receptor as a highly promising target for manipulating microglial innate immune response in AD and perhaps other neurodegenerative diseases. The mechanisms by which EP2-mediated signaling is related to Abeta phagocytosis and paracrine neurotoxicity are not known. Our Preliminary Data of mouse primary microglia expression identified a candidate gene that is tightly associated to EP2. We are aware of no report on the actions of this gene in any brain cells, including microglia. However, given emerging data we hypothesize that its expression and activity are mechanistically linked to EP2 signaling and that it may be a key element by which ablation of EP2 generated the highly desirable dual phenotype of enhanced Abeta phagocytosis and reduced paracrine neurotoxicity. We will test this hypothesis through the following Specific Aims: 1) Perform in vitro validation of our candidate mRNA and protein expression in wild type and EP2-/- primary mouse microglia before and after Abeta treatment. 2) Map the in vivo regional and cellular brain distribution in wild type mice and a transgenic mouse model of AD before and after following bone-marrow transplantation from either wild type or EP2-/- mice. 3) Determine the functionality of its expression in the context of the EP2-/- dual phenotype in both knockout mice and a microglial cell line using shRNA knockdown.