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.

项目摘要 周围神经损伤（PNI）引起运动神经元（MN）轴切开术，危害MN对细胞死亡的危害 在个人中，从来没有实现完整的功能恢复。因此，确定管理的机制 受伤或侮辱后的选择性MN损失对于MN疾病的治疗进步至关重要 改善患者的预后。研究表明，小胶质细胞被激活，增殖并迁移到受伤 PNI后的MN细胞体，显示了从促再生到退行性的一系列特征。现在， 尚不清楚的是用于生存或细胞死亡的受伤的MN上发生了哪些类型的小胶质细胞-MN相互作用。它 也未知小胶质细胞监测MN健康状况，这与MN命运的关系或哪种信号 机制调节了这些关系。我们的目标是识别小胶质动力学和细胞 与受伤的MN的通信，这些MN在PNI之后确定MN命运。我们将首先可视化小胶质细胞 使用高级显微镜方法的相互作用，然后探测可能调节的信号传导机制 各种健康状况的MN的小胶质细胞行为。菌落刺激因子1（CSF1），触发受体 在髓样细胞2（Trem2）和FC受体（FCR）上表达 通过12KDA（DAP12）和脾酪氨酸激酶（SYK）的DNAX激活蛋白的小胶质细胞激活 激活。文献表明，CSF1对于小胶质细胞增殖和向受伤的MN迁移是必要的。 初步数据表明，FCR在所有活化的小胶质细胞上均表示，而TREM2具有更大的表达 与存活或健康的MN相比，在垂死的MN周围的小胶质细胞上。鉴于先前的研究和 我们的初步发现，我预测小胶质细胞将显示不同的动态和吸收MN内容 取决于MN的健康状况以及保护环境之间的切换是 受DAP12和SYK协同激活程度的控制；更具体地说，TREM2正在行动 作为分子开关。为了进行测试，我将使用遗传的小胶质细胞使用PNI小鼠模型（坐骨轴切开术） 用GFP标记和轴向肌肉逆行的MNS标记。这将允许调查 在不同健康状况的受伤的MN上的小胶质细胞-MN相互作用。在AIM 1中，我将定量地表征 带有共聚焦，两光子和超分辨率的st型显微镜的小胶质细胞-MN动力学。这个目标将揭示 小胶质细胞如何从通过内吞或trogocytosis对MN含量进行采样转变为通过 神经吞噬作用。在AIM 2中，我将研究负责小胶质表型和的信号传导机制 它们随后对MN命运的影响。具体而言，我们将开发一个trem2小胶质细胞敲除小鼠模型 分析产生的小胶质细胞表型和MN相互作用，信号级联反应以及对MN的任何影响 再生或细胞死亡。这项研究将为PNI后神经保护干预提供基础。这 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。