Isolation of Terminal Schwann Cells by Fluorescence-Activated Cell Sorting

项目总结 脊椎动物的神经肌肉接头(NMJ)，像神经系统中的所有突触一样，具有 三个细胞成分：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(覆盖神经末梢的非髓鞘终末雪旺细胞(TSCS))。 在分子水平上，我们对TSCS知之甚少。然而，有证据表明，哺乳动物的TSCS 在成年动物神经损伤后突触连接的重建中发挥重要作用， 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)和脊肌萎缩症(SMA)。令人信服的是，这些都不是 研究直接测试了TSC是否对这些过程中的每一个都是必需的，以及机制是什么 可能是因为缺乏专门针对哺乳动物TSCS的遗传和分子工具。 这种工具的匮乏是由于缺乏经过验证的TSC特定标记。此R21应用程序提出了一种 使用荧光激活细胞分选(FACS)分离TSCs的创新方法将允许 以前所未有的规模鉴定TSC特异性标记。这种方法是基于比较的 FACS来源的髓鞘细胞和富含TSC的细胞库的RNA-Seq转录本 肌肉组织，在TSCs或髓鞘SCs中存在不同颜色的荧光报告。 这项工作的预期结果是鉴定出一组基本上完整的表达在 但在髓鞘干细胞中不存在。这些标记然后可以在未来的实验中使用，以选择性地 利用分子遗传学在体内操纵TSCs，从而更明确地确定它们对 正常和病理情况下的突触动态平衡。

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.