Microglial Activity on Injured Motoneurons

受损运动神经元的小胶质细胞活性

• 批准号: 10751692
• 负责人: TANA POTTORF
• 金额: $ 5.02万
• 依托单位: EMORY UNIVERSITY
• 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/10751692
• 关键词: 3-Dimensional; Amyotrophic Lateral Sclerosis; Axotomy; Behavior; Binding; Brachial plexus structure; CASP3 gene; Cd68; Cell Count; Cell Death; Cells; Central Nervous System; Cessation of life; Characteristics; Communication; Confocal Microscopy; Consumption; Cytoplasmic Granules; Data; Disease; Endocytosis; Environment; Excision; Fc Receptor; Filopodia; Foundations; Health Status; Histologic; Homeostasis; Image; Immune; In Situ Nick-End Labeling; Individual; Injury; Intervention; Investigation; Knockout Mice; Label; Life Experience; Literature; Lysosomes; Macrophage Colony-Stimulating Factor; Macrophage Colony-Stimulating Factor Receptor; Methods; Microglia; Microscopy; Modeling; Molecular; Monitor; Morphology; Motor; Motor Neurons; Mus; Muscle; Natural regeneration; Nerve Degeneration; Neuronal Injury; Neurons; Newborn Infant; Pathway interactions; Patient-Focused Outcomes; Peripheral nerve injury; Phagocytes; Phagocytosis; Phenotype; Phosphorylation; Process; Proliferating; Proteins; Recovery of Function; Research; Resolution; Risk; Role; SYK gene; Sampling; Signal Transduction; Slice; Spinal Cord; Stains; Surface; Surveys; TP53 gene; TREM2 gene; Testing; Therapeutic; Therapeutic Intervention; Time; Transfection; Visualization; Western Blotting; disability; glial activation; improved; injured; migration; mouse model; neuropathology; neuroprotection; particle; preservation; receptor; regenerative; response; superresolution microscopy; two-photon; ultra high resolution; uptake

Project Summary Peripheral nerve injuries (PNI) cause motoneuron (MN) axotomy, endangering MNs to cell death which results in individuals never achieving a full functional recovery. Therefore, identifying the mechanisms which govern selective MN loss following an injury or insult is crucial for therapeutic advancement in MN disease and for improving patient outcomes. Research has shown microglia become activated, proliferate and migrate to injured MN cell bodies after PNI, displaying a range of characteristics from pro-regenerative to degenerative. Currently, it is unknown what types of microglia-MN interactions occur on injured MNs destined for survival or cell death. It is also unknown how microglia monitor MN health status, how this relates to MN fate, or what signaling mechanisms regulate these relationships. Our objective is to identify microglial dynamics and cellular communication with injured MNs that determine MN fate following PNI. We will first visualize microglia-MN interactions using advanced microscopy methods and then probe for signaling mechanisms that may regulate microglia behavior on MNs of various health statuses. Colony Stimulating Factor 1 (CSF1), Triggering Receptor Expressed on Myeloid Cells 2 (TREM2), and Fc Receptors (FcR) have all been independently shown to modulate microglial activation via DNAX activating protein of 12kDa (DAP12) and Spleen Tyrosine Kinase (SYK) activation. Literature has shown CSF1 is necessary for microglia proliferation and migration towards injured MNs. Preliminary data shows FcRs are expressed on all activated microglia, whereas TREM2 has a greater expression on microglia surrounding dying MNs compared to surviving or healthy MNs. Given the previous research and our preliminary findings, I predict that microglia will display different dynamics and uptake of MN content depending on the MN health status and that the switch between protective to death environments is governed by the degree of synergistic activation of DAP12 and SYK; more specifically that TREM2 is acting as a molecular switch. To test this, I will use a PNI mouse model (sciatic axotomy) with microglia genetically labeled with GFP and axotomized MNs retrogradely labeled from muscle. This will allow the investigation of microglial-MN interactions on injured MNs of different health statuses. In aim 1, I will quantitatively characterize microglia-MN dynamics with confocal, two-photon, and super-resolution STED microscopy. This aim will reveal how microglia could change from sampling MN contents through endocytosis or trogocytosis to MN removal by neuronal phagocytosis. In aim 2, I will investigate signaling mechanisms that govern microglial phenotypes and their subsequent effect on MN fate. Specifically, we will develop a TREM2 microglia knock-out mouse model to analyze resulting microglia phenotypes and MN interactions, signaling cascades, and any effects on MN regeneration or cell death. This research will provide foundations for neuroprotective interventions after PNI. The signaling and cellular interactions revealed could be transferable to other neuropathologies.

项目概要 周围神经损伤 (PNI) 会导致运动神经元 (MN) 轴索切断，危及 MN 细胞死亡，从而导致细胞死亡 在从未实现完全功能恢复的个体中。因此，确定控制机制 损伤或侮辱后选择性 MN 丧失对于 MN 疾病的治疗进展和 改善患者的治疗效果。研究表明小胶质细胞被激活、增殖并迁移到受伤部位 PNI 后的 MN 细胞体表现出从促再生到退化的一系列特征。现在， 目前尚不清楚在注定要生存或细胞死亡的受损 MN 上会发生什么类型的小胶质细胞-MN 相互作用。它 还不清楚小胶质细胞如何监测 MN 的健康状况，这与 MN 的命运有何关系，或者发出什么信号 机制调节这些关系。我们的目标是确定小胶质细胞动力学和细胞 与受伤的 MN 进行通信，决定 PNI 后 MN 的命运。我们首先将小胶质细胞-MN 可视化 使用先进的显微镜方法相互作用，然后探索可能调节的信号机制 不同健康状态的 MN 上的小胶质细胞行为。集落刺激因子 1 (CSF1)，触发受体 在骨髓细胞 2 (TREM2) 和 Fc 受体 (FcR) 上表达均已被独立证明可以调节 通过 12kDa (DAP12) 的 DNAX 激活蛋白和脾酪氨酸激酶 (SYK) 激活小胶质细胞 激活。文献表明 CSF1 对于小胶质细胞增殖和向受伤的 MN 迁移是必需的。 初步数据显示，FcR 在所有激活的小胶质细胞上都有表达，而 TREM2 的表达量更高 与幸存或健康的 MN 相比，垂死 MN 周围的小胶质细胞的变化。鉴于之前的研究和 我们的初步发现，我预测小胶质细胞将表现出不同的动态和对 MN 内容的摄取 取决于 MN 的健康状况，并且保护环境与死亡环境之间的切换是 受 DAP12 和 SYK 协同激活程度的控制；更具体地说，TREM2 正在发挥作用 作为分子开关。为了测试这一点，我将使用带有小胶质细胞基因的 PNI 小鼠模型（坐骨神经轴索切除术） 用 GFP 标记，并从肌肉逆行标记轴切 MN。这将允许调查 小胶质细胞-MN 对不同健康状况的受损 MN 的相互作用。在目标 1 中，我将定量描述 使用共焦、双光子和超分辨率 STED 显微镜观察小胶质细胞-MN 动力学。这个目标将揭示 小胶质细胞如何从通过内吞作用或吞噬作用采样 MN 内容，转变为通过胞吞作用或吞噬作用去除 MN 神经元的吞噬作用。在目标 2 中，我将研究控制小胶质细胞表型和 它们对 MN 命运的后续影响。具体来说，我们将开发 TREM2 小胶质细胞敲除小鼠模型 分析由此产生的小胶质细胞表型和 MN 相互作用、信号级联以及对 MN 的任何影响 再生或细胞死亡。这项研究将为 PNI 后的神经保护干预提供基础。这 所揭示的信号传导和细胞相互作用可以转移到其他神经病理学。