Circadian Control of Heparan Sulfate Levels Times Phagocytosis of Amyloid Beta Aggregates

The following datasets are from the manuscript entitled “Circadian Control of Heparan Sulfate Levels Times Phagocytosis of Amyloid Beta Aggregates” published in PLOS Genetics. This paper investigates the link between Alzheimer’s disease and circadian disruption by looking at naïve macrophages and the phagocytosis of a neurotoxic protein involved in Alzheimer’s Disease, amyloid-beta (1-42). We found that there is a circadian oscillation in the phagocytosis of amyloid-beta (1-42) with the peak of phagocytosis at Post Shock time (PS) 32 and the trough at PS 16. To confirm the observed rhythm, we repeated our phagocytosis experiment in PER1-/-/PER2-/- knockout cells, which lack a circadian rhythm, and observed a change in the rhythm, confirming circadian influence. We then probed a previously published macrophage proteomic dataset (Collins et al., Genome Research, 2021) and found genes involved in the heparan sulfate and chondroitin sulfate biosynthesis pathways to be circadianly controlled. To demonstrate the circadian control of cells surface heparan sulfate and chondroitin sulfate proteoglycans, we performed liquid chromatography tandem mass spectrometry on our macrophages and found that there is a circadian rhythm to both heparan sulfate and chondroitin sulfate proteoglycan expression. We found that the rhythm of heparan sulfate expression was anti-phase to the oscillation of amyloid-beta phagocytosis, indicating an inhibitory relationship. Therefore, we performed phagocytosis experiments in the presence of heparan sulfate cleaving enzymes (heparinases I, II, and III) and found an ablation of the previously observed rhythm of amyloid-beta phagocytosis. Furthermore, we investigated if this oscillation was unique to amyloid-beta (1-42) by repeating our phagocytosis experiments with other homologs of amyloid-beta, amyloid-beta (1-40) and mouse amyloid-beta (1-42). These homologs were of interest as they do not rapidly aggregate like amyloid-beta (1-42) and mouse amyloid-beta contains mutations in the known heparan sulfate proteoglycan binding region. We found that the amyloid-beta homologs did not have an oscillation in phagocytosis, demonstrating that aggregation and heparan sulfate proteoglycan binding are essential to the observed rhythm. The attached datasets are from all the above-mentioned experiments. For the phagocytosis experiments, the fluorescent microscopy images as well as the determined pixel intensity data are included. For the liquid chromatography tandem mass spectrometry data, the normalization method as well as the raw and normalized data are available.本数据集来源于发表于《公共科学图书馆·遗传学》（PLOS Genetics）的学术论文《硫酸乙酰肝素水平的昼夜节律调控介导β淀粉样蛋白聚集的吞噬时序》。该研究围绕阿尔茨海默病（Alzheimer’s Disease, AD）与昼夜节律紊乱的关联展开，以未致敏巨噬细胞（naïve macrophages）为研究模型，聚焦阿尔茨海默病相关神经毒性蛋白β淀粉样蛋白1-42（amyloid-beta (1-42)）的吞噬过程。研究发现，β淀粉样蛋白1-42的吞噬活性存在昼夜节律振荡，其吞噬峰值出现在震后时间（Post Shock time, PS）32时，谷值则位于PS 16时。 为验证所观测到的节律，我们在缺失昼夜节律的PER1基因及PER2基因双敲除细胞（PER1-/-/PER2-/- knockout cells）中重复了吞噬实验，结果观测到该节律发生显著改变，证实了昼夜节律对吞噬过程的调控作用。随后，我们检索了已发表的巨噬细胞蛋白质组数据集（Collins等，《基因组研究》（Genome Research），2021年），发现参与硫酸乙酰肝素（heparan sulfate）与硫酸软骨素（chondroitin sulfate）生物合成通路的基因均受昼夜节律调控。 为验证细胞表面硫酸乙酰肝素蛋白聚糖与硫酸软骨素蛋白聚糖的昼夜节律调控特性，我们对巨噬细胞进行了液相色谱-串联质谱（liquid chromatography tandem mass spectrometry, LC-MS/MS）分析，结果显示两类蛋白聚糖的表达均存在昼夜节律。研究发现，硫酸乙酰肝素的表达节律与β淀粉样蛋白吞噬振荡呈反相，提示二者存在抑制性关系。基于此，我们在硫酸乙酰肝素裂解酶（即肝素酶I、II、III（heparinases I, II, III））存在的条件下开展吞噬实验，结果发现此前观测到的β淀粉样蛋白吞噬节律被完全消除。 此外，我们通过使用β淀粉样蛋白的其他同源亚型——β淀粉样蛋白1-40（amyloid-beta (1-40)）与小鼠源β淀粉样蛋白1-42（mouse amyloid-beta (1-42)）重复吞噬实验，探究该振荡是否为β淀粉样蛋白1-42所特有。选择上述同源亚型开展实验的原因在于，它们不会像β淀粉样蛋白1-42那样快速聚集，且小鼠源β淀粉样蛋白在已知的硫酸乙酰肝素蛋白聚糖结合区域存在氨基酸突变。实验结果显示，这些β淀粉样蛋白同源亚型的吞噬过程均未出现节律振荡，证实了蛋白聚集与硫酸乙酰肝素蛋白聚糖结合是观测到该节律的必要条件。 本附件数据集来源于上述全部实验。其中，吞噬实验相关数据包含荧光显微镜图像与测定的像素强度数据；液相色谱-串联质谱数据则包含标准化方法、原始数据与标准化后的数据。