Bradykinin promotes immune responses in differentiated embryonic neurospheres carrying APP swe and PS1 dE9 mutations. Bradykinin promotes immune responses in differentiated embryonic neurospheres carrying APP swe and PS1 dE9 mutations

Neural stem cells (NSCs) can be cultivated from developing brains, reproducing many of the processes that occur during neural development. They can be isolated from a variety of animal models, such as transgenic mice carrying mutations in amyloid precursor protein (APP) and presenilin 1 and 2 (PSEN 1 and 2), characteristic of familial Alzheimer’s Disease (AD). Modulating the development of these cells with inflammation-related peptides, like bradykinin (BK) and its antagonist HOE-140, enables the understanding of the impact of such molecules on the genetic load of treated neurospheres. To facilitate an understanding of the wider biological processes in the context of neuroinflammation, we performed a global gene expression analysis on the transgenic neurospheres treated with BK and HOE-140. To validate the microarray data, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed on 8 important genes related to the immune response in AD. We then performed a comparative analysis of the transcriptional profiles between treatments, including gene ontology and reactome enrichment, construction and analysis of protein-protein interaction networks and, finally, comparing our data with human datasetd from AD patients. We observed that the treatments modulated the expression of genes mainly related to neuroinflammation mediated by microglia, with BK promoting an increase in the expression of genes that enrich processes, pathways, and cellular components related to immune impairment, neurodegeneration and cell cycle. B2 receptor inhibition by HOE-140 acting on reduction of AD-related anomalies caused in this system. Our results reinforce that BK is an important modulating agent and enhances the immunological changes identified in transgenic neurospheres that carry the genetic load of AD. Overall design: Four condition experiment, APPswe/PS1dE9 (APP_WT) vs. WT cells, APPswe/PS1dE9 vs. APPswe/PS1dE9 treated with BK (BK_APP) and APPswe/PS1dE9 vs. APPswe/PS1dE9 treated with HOE-140 (HOE_APP). Biological replicates: 4 APP, 4 WT, 4 BK replicates, 4 HOE-140.

神经干细胞（Neural stem cells, NSCs）可从发育中的脑组织中培养获得，能够重现神经发育过程中的诸多生物学进程。该类细胞可从多种动物模型中分离得到，例如携带淀粉样前体蛋白（amyloid precursor protein, APP）以及早老素1、2（presenilin 1 and 2, PSEN 1和2）突变的转基因小鼠——这两类突变是家族性阿尔茨海默病（familial Alzheimer’s Disease, AD）的典型特征。通过缓激肽（bradykinin, BK）及其拮抗剂HOE-140等炎症相关肽类调控此类细胞的发育，可用于解析此类分子对经处理的神经球遗传负荷的影响。为便于解析神经炎症情境下的更广范围生物学进程，我们对经BK与HOE-140处理的转基因神经球开展了全局基因表达分析。为验证微阵列数据，我们针对8个与阿尔茨海默病免疫应答相关的关键基因，开展了实时定量反转录聚合酶链式反应（quantitative real-time reverse-transcription polymerase chain reaction, RT-PCR）实验。随后我们对各处理组的转录谱进行了比较分析，涵盖基因本体论（gene ontology）富集分析、Reactome通路富集分析、蛋白质相互作用网络的构建与分析，并最终将本研究数据与阿尔茨海默病患者的人类数据集进行了比对。我们观察到，各处理组主要调控了小胶质细胞介导的神经炎症相关基因的表达：BK可促进与免疫损伤、神经退行性变及细胞周期相关的进程、通路及细胞组分富集的基因表达上调；而HOE-140通过抑制缓激肽B2受体，可减轻本系统中出现的阿尔茨海默病相关异常。本研究结果进一步证实，BK是重要的调控因子，可增强携带阿尔茨海默病遗传负荷的转基因神经球中观测到的免疫变化。整体实验设计：本研究包含四组条件的对照实验，分别为APPswe/PS1dE9（APP_WT）细胞与野生型（WT）细胞、经BK处理的APPswe/PS1dE9（BK_APP）细胞与APPswe/PS1dE9细胞、经HOE-140处理的APPswe/PS1dE9（HOE_APP）细胞与APPswe/PS1dE9细胞。生物学重复设置为：APP组、WT组、BK处理组及HOE-140处理组各4例。