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无法消化的物体的过程。 被吞噬。我们称这个过程为消化外食，并且我们已经证明了它在消化中的作用 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食来修剪和部分降解 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。消化性外食涉及 在待降解物体（溶酶体突触）上形成紧密密封，分泌晚期内体 和溶酶体 (LE/Ly) 进入溶酶体突触，并通过 V-ATP 酶质子泵酸化。我们有 有证据表明培养中的原代小胶质细胞在与β-淀粉样蛋白接触后形成溶酶体突触。在 目标 1 我们将继续描述这一点。首先，我们将确认小胶质细胞产生的初步数据 当它们与β-淀粉样蛋白相互作用时，酸化的、富含F-肌动蛋白的接触区域会分泌溶酶体 内容到这些领域。然后我们将量化这些中淀粉样蛋白的时间依赖性降解 隔间。我们将确定抗 Aβ IgG 是否会增加小胶质细胞对斑块的消化。我们将 还使用光学和电子显微镜检查 AD 模型小鼠消化外食的证据。 我们通过以下方法部分表征了消化外食所需的信号机制 巨噬细胞，我们将用它作为小胶质细胞研究的模型。我们注意到信号通路 我们描述了消化外食的遗传研究中涉及的重叠信号通路 阿尔茨海默病。在目标 2 中，我们将探讨其中一些突变对消化性外食的影响。 这些包括 Trem2R47H、Dap12-/- 和 Trem2-/- 小鼠小胶质细胞。我们已经证明药理学 操纵 PI3 激酶和 Akt 以及其他信号分子可改变消化外噬 巨噬细胞，我们将扩展这些药物对消化系统影响的初步研究 小胶质细胞对淀粉样蛋白的外噬。 小胶质细胞被认为在神经原纤维（包括 tau 蛋白和 tau 蛋白）的传播中发挥作用。 未消化的 Aβ 原纤维。 LE/Ly 分泌是消化外食的重要组成部分，可能 有助于部分消化的 tau 丝或 Aβ 原纤维片段从小胶质细胞中扩散出来， 我们将在第三个目标中研究这一点。具体来说，我们将确定增强的 LE/Ly 是否 当小胶质细胞接触β-淀粉样蛋白时，其分泌会导致先前内吞的原纤维的分泌增强。 然后，我们将测试目标 1 和 2 中描述的治疗对原纤维内吞作用和消化的影响 它们在与淀粉样蛋白接触后分泌。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 衍生的小胶质细胞。