Isolation of Terminal Schwann Cells by Fluorescence-Activated Cell Sorting

项目摘要 脊椎动物神经肌肉接头（NMJ），像整个神经系统的所有突触一样， 三种细胞成分：突触前细胞（运动神经元）、突触后细胞（骨骼肌 纤维）和神经胶质包裹物（覆盖神经末梢的无髓鞘终末许旺细胞（tSC））。 在分子水平上，我们对tSC的了解最少。然而，有证据表明，哺乳动物的tSC 在成年动物神经损伤后突触连接的重建中起重要作用， 调节新生儿的突触修剪，并可能在神经肌肉疾病的早期阶段发挥关键作用 肌萎缩侧索硬化症（ALS）和脊髓性肌萎缩症（SMA）。令人信服的，因为他们不是这些 研究直接测试了tSC是否对这些过程中的每一个都是必要的，以及什么机制是必要的。 这可能是因为缺乏特异性靶向哺乳动物tSC的遗传和分子工具。 这种工具的稀缺性是由于缺乏已证实的TSC特异性标记物。本R21申请提出了一种 使用荧光激活细胞分选（FACS）分离tSC的创新方法， 以前所未有的更大规模鉴定TSC特异性标志物。该方法基于比较 通过流式细胞术（FACS）获得的髓鞘形成和TSC富集细胞池的RNA-Seq生成的转录组来自 肌肉组织，其中不同颜色的荧光报告基因存在于tSC或髓鞘形成SC中。 这项工作的预期成果是鉴定出在大肠杆菌中表达的一组基本完整的基因。 tSC中，但不在髓鞘形成SC中。这些标记物可以在未来的实验中选择性地 使用分子遗传学在体内操纵tSC，从而更明确地确定它们对 正常和病理情况下的突触稳态。

PROJECT SUMMARY The vertebrate neuromuscular junction (NMJ), like all synapses throughout the nervous system, has three cellular components: the presynaptic cell (the motor neuron), the postsynaptic cell (the skeletal muscle fiber) and the glial wrappings (the nonmyelinating terminal Schwann cells (tSCs) that cap the nerve terminal). At the molecular level, we know the least about tSCs. Nevertheless, evidence suggests that mammalian tSCs play important roles in re-establishment of synaptic connections following nerve damage in adult animals, regulate synapse pruning in neonates, and may have key roles at early stages of the neuromuscular diseases amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Compelling as they are none of these studies directly tested whether tSC are necessary for each of these processes and what mechanisms are possibly involved because there is a lack of genetic and molecular tools specifically targeting mammalian tSCs. This scarcity of tools is due to the paucity of proven tSC-specific markers. This R21 application proposes an innovative method for isolating tSCs using fluorescence-activated cell sorting (FACS) that will allow identification of tSC-specific markers at an unprecedented larger scale. The approach is based on comparing the RNA-Seq-generated transcriptomes of myelinating and tSC-enriched cell pools derived by FACS from muscle tissue in which fluorescent reporters of different colors are present in either tSCs or myelinating SCs. The expected outcome of this work is the identification of a largely complete set of those genes expressed in tSCs but not in myelinating SCs. These markers then could be used in future experiments to selectively manipulate tSCs in vivo using molecular genetics and thus determine more definitely their contribution to synaptic homeostasis in normal and pathological situations.