Role of microglial lysosomes in amyloid-A-beta degradation

小胶质细胞溶酶体在淀粉样蛋白-A-β降解中的作用

• 批准号: 10734289
• 负责人: Frederick R. Maxfield
• 金额: $ 168.47万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10734289
• 关键词: Actins; Adipocytes; Affect; Alzheimer' s Disease; Alzheimer' s disease model; Amyloid; Amyloid beta-Protein; Animals; Antibodies; Area; Atherosclerosis; Binding; Biological Assay; Brain; Cell Culture Techniques; Cells; Confocal Microscopy; Cultured Cells; Data; Deposition; Development; Digestion; Electron Microscopy; Endocytosis; F-Actin; Fluorescence; Genetic study; Goals; Human; Hydrolysis; Immune; Immunoglobulin G; Lipoproteins; Low-Density Lipoproteins; Lysosomes; MFAP1 gene; Macrophage; Measures; Methods; Microglia; Modeling; Mus; Mutant Strains Mice; Mutation; Neuropathy; Optics; PIK3CG gene; Pathway interactions; Phagocytes; Pharmaceutical Preparations; Phosphatidylinositide 3-Kinase Inhibitor; Play; Polymers; Process; Proteins; Proton Pump; Role; Senile Plaques; Signal Pathway; Signal Transduction; Signaling Molecule; Study models; Synapses; TREM2 gene; Testing; Time; Wild Type Mouse; animal tissue; extracellular; genetic testing; in vivo; induced pluripotent stem cell; late endosome; mouse model; multiphoton microscopy; novel; pharmacologic; polymerization; seal; tau Proteins; tau aggregation; tissue culture; treatment effect; uptake; vacuolar H+-ATPase

We have characterized a process used by phagocytic cells to digest objects that are too large to be phagocytosed. We call this process digestive exophagy, and we have demonstrated its role in digestion of large aggregates of lipoproteins in atherosclerosis and also in the digestion of dead adipocytes. We hypothesize that microglia use digestive exophagy to trim and partially degrade β-amyloid. This may be the mechanism by which microglia limit the expansion of β-amyloid plaques. Digestive exophagy involves the formation of a tight seal on the object to be degraded (a lysosomal synapse), secretion of late endosomes and lysosomes (LE/Ly) into the lysosomal synapse, and acidification by V-ATPase proton pumps. We have evidence that primary microglia in culture form lysosomal synapses upon contact with β-amyloid. In Aim 1 we will continue to characterize this. First, we will confirm our preliminary data that microglia create acidified, F-actin-rich contact regions when they interact with β-amyloid and that they secrete lysosomal contents into these areas. We will then quantify time-dependent degradation of amyloid in these compartments. We will determine if anti- Aβ IgG increases digestion of the plaques by microglia. We will also use optical and electron microscopy to examine evidence for digestive exophagy in AD model mice. We have partially characterized the signaling mechanisms required for digestive exophagy by macrophages, and we will use this as a model for studies in microglia. We note that the signaling pathways we described for digestive exophagy overlap signaling pathways implicated in genetic studies of Alzheimer’s disease. In Aim 2 we will explore the effects of some of these mutations on digestive exophagy. These will include Trem2R47H, Dap12-/-, and Trem2-/- mouse microglia. We have shown that pharmacological manipulation of PI3-kinase and Akt as well as other signaling molecules alter digestive exophagy in macrophages, and we will extend our preliminary studies on the effects of these drugs on digestive exophagy of amyloid by microglia. Microglia have been proposed to play a role in the spread of neuropathic fibrils, including tau and undigested Aβ fibrils. LE/Ly secretion, which is an essential component of digestive exophagy, may contribute to the spread of partially digested fragments of tau filaments or Aβ fibrils out of microglial cells, and we will examine this in the third Aim. Specifically, we will determine whether the enhanced LE/Ly secretion when microglia contact β-amyloid leads to enhanced secretion of previously endocytosed fibrils. We will then test the effects of treatments described in Aims 1&2 on endocytosis and digestion of fibrils and their secretion upon contact with amyloid. In our tissue culture studies, we will use primary mouse microglia and human iPSC-derived microglial cells.

我们已经描述了吞噬细胞用来消化太大而不能 被吞噬了。我们称这一过程为消化外噬，我们已经证明了它在消化 动脉粥样硬化和死亡脂肪细胞消化过程中脂蛋白的大量聚集。我们 假设小胶质细胞利用消化外噬作用来修剪和部分降解β-淀粉样蛋白。这可能是 小胶质细胞限制β-淀粉样斑块扩张的机制。消化性外噬涉及到 在被降解的物体(溶酶体突触)上形成紧密的密封，分泌晚期内小体 和溶酶体(LE/Ly)进入溶酶体突触，并被V-ATPase质子泵酸化。我们有 培养的原代小胶质细胞与β-淀粉样蛋白接触后形成溶酶体突触的证据。在……里面 目标1我们将继续描述这一点。首先，我们将确认我们的初步数据，小胶质细胞创建 当它们与β-淀粉样蛋白相互作用时，酸性富含F-肌动蛋白的接触区和它们分泌溶酶体 内容进入这些区域。然后我们将量化这些淀粉样蛋白随时间的降解 车厢。我们将确定抗Aβ免疫球蛋白是否能增加小胶质细胞对斑块的消化。我们会 还使用光学和电子显微镜来检查AD模型小鼠消化外噬的证据。 我们已经部分地描述了消化外噬所需的信号机制 巨噬细胞，我们将用它作为研究小胶质细胞的模型。我们注意到，信号通路 我们描述了消化性外噬重叠信号通路在遗传学研究中的作用 阿尔茨海默氏症。在目标2中，我们将探索这些突变中的一些对消化外噬的影响。 这些将包括Trem2R47H、Dap12-/-和TREM2-/-小鼠小胶质细胞。我们已经证明了药理作用 PI3-激酶和Akt等信号分子的调控改变消化系统的外噬作用 巨噬细胞，我们将扩大我们对这些药物对消化系统影响的初步研究 小胶质细胞吞噬淀粉样蛋白。 小胶质细胞被认为在神经病理性纤维的扩散中发挥作用，包括tau和 未消化的Aβ纤维。LE/Ly分泌是消化性外噬作用的重要组成部分，它可能 有助于将部分消化的tau细丝或Aβ纤维片段从小胶质细胞扩散出去， 我们将在第三个目标中检查这一点。具体来说，我们将确定增强的LE/LY 小胶质细胞接触β-淀粉样蛋白时的分泌导致先前内吞的纤维分泌增加。 然后，我们将测试目标1和目标2中描述的处理对纤维和纤维的内吞和消化的影响 它们与淀粉样蛋白接触时的分泌物。在我们的组织培养研究中，我们将使用原代小鼠小胶质细胞 和人IPSC来源的小胶质细胞。