Microglial Activity on Injured Motoneurons

项目摘要 周围神经损伤（PNI）引起运动神经元（MN）轴突切断，危及MN细胞死亡， 无法完全恢复功能的人因此，确定管理的机制 损伤或损伤后的选择性MN损失对于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，增殖和迁移到受伤的 MN细胞体PNI后，显示出一系列的特点，从促再生退化。目前， 在注定存活或细胞死亡的受损MN上发生何种类型的小胶质细胞-MN相互作用是未知的。它 也不知道小胶质细胞如何监测MN的健康状况，这与MN的命运如何相关，或者是什么信号 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 与确定PNI后MN命运的受伤MN通信。我们首先将小胶质细胞MN 使用先进的显微镜方法进行相互作用，然后探测可能调节 小胶质细胞的行为对各种健康状况的MN。集落刺激因子1（CSF 1），触发受体 髓样细胞2（TREM 2）和Fc受体（FcR）都已独立地显示出调节 通过DNAX激活蛋白12 kDa（DAP 12）和脾酪氨酸激酶（SYK）激活小胶质细胞 activation.文献表明，CSF 1是小胶质细胞增殖和向受损MN迁移所必需的。 初步数据显示，FcRs在所有活化的小胶质细胞上表达，而TREM 2在活化的小胶质细胞上表达更高。 与存活或健康的MN相比，对死亡MN周围的小胶质细胞的影响。根据之前的研究， 根据我们的初步发现，我预测小胶质细胞将显示不同的动力学和MN含量的摄取， 取决于MN的健康状况，并且在保护性环境与死亡环境之间的切换是 由DAP 12和SYK的协同激活程度控制;更具体地说，TREM 2是起作用的。 as a molecular分子switch开关.为了验证这一点，我将使用一个PNI小鼠模型（坐骨神经切断术）与小胶质细胞遗传 标记的GFP和从肌肉逆行标记的轴突切断的MN。这将有助于调查 小胶质细胞-MN相互作用对不同健康状态的受损MN的影响。在目标1中，我将定量描述 小胶质细胞-MN动力学与共焦，双光子，和超分辨率STED显微镜。这一目标将揭示 小胶质细胞如何从通过内吞作用或胞吞作用取样MN含量到通过 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的信号传导机制， 其对MN命运的后续影响。具体来说，我们将开发一种TREM 2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，以及对MN的任何影响 再生或细胞死亡。本研究将为PNI后神经保护干预提供依据。的 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。

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.