Macrophage-lipoprotein Interactions

摘要 我们已经描述了一个由吞噬细胞进行的新过程，我们称之为“消化”过程。 外食”。我们已经证明，巨噬细胞和树突状细胞产生细胞外，酸化溶酶体， 隔室消化大的物体，如LDL或死亡的脂肪细胞的聚集体，太大而不能被消化。 被吞噬我们已经发现与神经退行性疾病相关的基因突变（例如， 额颞叶痴呆症和阿尔茨海默氏病）也影响聚集LDL的消化外食 （agLDL），如在动脉粥样硬化斑块中发现的。这与类似的细胞机制是一致的 参与了这两起案件在本附录中，我们建议将我们开发的方法应用于 用小胶质细胞研究淀粉样蛋白Aβ消化性外噬我们还将继续研究 神经变性的研究，其对巨噬细胞消化agLDL的影响。我们已经报道了 缺乏颗粒蛋白前体（GRN）的巨噬细胞对聚集的LDL的外噬作用大大增加。突变或 GRN的表达减少与AD以及额颞叶痴呆有关。TLR4和其他信号传导 分子是消化性外食所必需的，这些相同的信号传导途径由 TREM2是一种在小胶质细胞中高度表达的蛋白质。TREM2的突变与增加的 AD的易感性。 初步研究表明，小胶质细胞在接触到 fAβ的大聚集体，并向其中分泌溶酶体内容物。在目标1中，我们将描述 小胶质细胞降解fAβ大聚集体的机制。我们希望初步确认 溶酶体分泌和隔室酸化的观察。我们将使用荧光标记的fAβ， 观察并量化fAβ沉积物的降解。这些研究将使用主要的 小鼠小胶质细胞。在目标2中，我们将分析调节溶酶体分泌的信号机制 以及小胶质细胞对淀粉样蛋白Aβ的降解。有趣的是，初步数据显示，缺乏 骨髓巨噬细胞中TREM2信号的效应子Dap12导致溶酶体大量增加 与LDL聚集体或Aβ沉积物接触后分泌。类似的研究将在小胶质细胞中进行 从接触Aβ沉积物的野生型和基因敲除小鼠中，以确定GRN，Trem2， Dap12、Tlr4和其他蛋白质。如Dap12结果所示，在阿尔茨海默氏症中鉴定的蛋白质 研究可能揭示了巨噬细胞中新的信号传导，将在母基金中进行研究。

Abstract We have characterized a novel process carried out by phagocytes that we have called "digestive exophagy". We have shown that macrophages and dendritic cells create extracellular, acidified lysosomal compartments to digest large objects such as aggregates of LDL or dead adipocytes that are too large to be phagocytosed. We have found that genetic mutations associated with neurodegenerative disorders (e.g., frontotemporal dementia and Alzheimer's disease) also affect the digestive exophagy of aggregated LDL (agLDL), such as that found in atherosclerotic plaques. This is consistent with similar cellular mechanisms being involved in both cases. In this supplement, we propose to apply the methods we have developed to study digestive exophagy of amyloid Aβ by microglia. We will also continue to examine mutants identified in neurodegeneration studies for their effect on digestion of agLDL by macrophages. We have reported that macrophages lacking progranulin (GRN) have greatly increased exophagy of aggregated LDL. Mutations or reduced expression of GRN are linked to AD as well as frontotemporal dementia. TLR4 and other signaling molecules are required for digestive exophagy, and these same signaling pathways are modulated by TREM2, a protein that is highly expressed in microglia. Mutations in TREM2 are strongly linked to increased susceptibility for AD. Preliminary studies have shown that microglia create similar acidic compartments upon contact with large aggregates of fAβ, and they secrete lysosomal contents into them. In Aim 1 we will characterize the mechanism by which microglia degrade large aggregates of fAβ. We expect to confirm preliminary observations of lysosome secretion and compartment acidification. We will use fluorescently labeled fAβ to observe and quantify the degradation of fAβ deposits. These studies will be carried out using primary mouse microglia. In Aim 2 we will analyze signaling mechanisms that regulate lysosome secretion and degradation of amyloid Aβ by microglia. Interestingly, preliminary data have shown that lack of Dap12, an effector of TREM2 signaling, in bone marrow macrophages leads to a large increase in lysosome secretion upon contact with LDL aggregates or Aβ deposits. Similar studies will be carried out in microglia from wild type and knockout mice contacting Aβ deposits to determine if there is a role for GRN, Trem2, Dap12, Tlr4 and other proteins. As illustrated by the Dap12 results, proteins identified in the Alzheimer's studies may reveal new signaling in macrophages to be studied in the parent grant.