Microglial Activity on Injured Motoneurons

项目摘要 周围神经损伤(PNI)导致运动神经元(MN)轴突切断，危及MN的细胞死亡 在个体中永远不会实现完全的功能恢复。因此，确定治理的机制 损伤或侮辱后的选择性MN丢失对于MN疾病的治疗进展和 改善患者的预后。研究表明，小胶质细胞被激活、增殖并迁移到损伤处 PNI后的MN细胞体，表现出从促再生到退化的一系列特征。目前， 目前尚不清楚在受损的MN上发生了何种类型的小胶质细胞-MN相互作用，最终导致了生存或细胞死亡。它 也不知道小胶质细胞如何监控MN的健康状态，这与MN的命运有什么关系，或者是什么信号 机制调节着这些关系。我们的目标是识别小胶质细胞动力学和细胞 与受伤的MN沟通，决定PNI后MN的命运。我们将首先可视化小胶质细胞-MN 使用先进的显微镜方法相互作用，然后探测可能调节的信号机制 小胶质细胞在不同健康状态的MNS上的行为。激活受体的集落刺激因子1(CSF1) 在髓样细胞2(TREM2)和Fc受体(FCR)上的表达都被证明是独立地调节 12 kDa DNAX激活蛋白(DAP12)和脾酪氨酸激酶(SYK)激活小胶质细胞 激活。文献表明，CSF1是小胶质细胞增殖和向受损的MNS迁移所必需的。 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的表达更强 在死亡的MN周围的小胶质细胞上，与存活或健康的MN相比。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力学和摄取MN含量 取决于MN的健康状态以及从保护环境到死亡环境的切换 受DAP12和SYK协同激活的程度控制；更具体地说，是TREM2起作用 作为一个分子开关。为了测试这一点，我将使用带有小胶质细胞的PNI小鼠模型(坐骨神经轴突切断术) GFP标记和肌肉逆行标记的切断轴突的MN。这将允许调查 不同健康状态受损MN的小胶质细胞-MN相互作用。在目标1中，我将定量描述 用共聚焦、双光子和超分辨率STED显微镜观察小胶质细胞-MN动力学。这一目标将揭示 小胶质细胞如何通过内吞或巨噬细胞吞噬来采集MN含量，并通过 神经元吞噬作用。在目标2中，我将研究控制小胶质细胞表型和 他们随后对MN命运的影响。具体地说，我们将开发一种TREM2小胶质细胞敲除小鼠模型来 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。本研究将为PNI后的神经保护干预提供依据。这个 揭示的信号和细胞相互作用可能会转移到其他神经病理中。

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.