Single cell transcriptomics of nerves that lack Remak bundles

项目摘要 雪旺细胞(Schwann cell，SCs)是一种来源于神经沟的细胞，其主要功能是支持和保护 周围神经系统中的神经元。干细胞可分为两种亚型：有髓鞘的和无髓鞘的 髓鞘形成。髓鞘干细胞与神经元形成1：1的关系，并将它们包裹在髓鞘中，以提供快速的 神经冲动的传导，如传递本体感觉和触觉所需的神经冲动。相比之下， 非髓鞘干细胞通常将多个轴突捆绑成称为Remak束的神经结构， 支持电池，但不加快传输速度。干细胞功能障碍的个体表现出各种不同的 表型可能包括感觉减退、虚弱和疼痛，这些疾病属于 人类最常见的遗传性疾病。此外，干细胞也是肿瘤发生的靶细胞类型 在神经纤维瘤综合征(NF1和NF2)中，NF1和NF2与 周围神经和其他部位。Rac1是Rho家族的一种小的GTPase蛋白，它已经被 在NF1和NF2相关肿瘤以及其他形式的遗传性肿瘤中，被证明是肿瘤发生所必需的 雪旺细胞瘤(如卡尼复合体)。最近，对遗传性SC功能障碍综合征的分析已经 也显示了rac1的调节失调。为了研究rac1信号在干细胞功能和肿瘤发生中的作用， 我的实验室已经产生了一个携带有条件激活的等位基因rac1的小鼠模型，并进行了 激活rac1在干细胞发育不同阶段的作用的初步表征。我们观察到 在早期胚胎发生中，rac1的激活促进了细胞的增殖，并影响了非 髓鞘干细胞形成正常的Remak束，在rac1激活的情况下，这些束从神经中完全消失 在早期胚胎发育中。在这个R03应用程序中，我们假设rac1激活会导致异常 在不影响髓鞘干细胞轨迹的情况下，分化为非髓鞘干细胞。首字母 通过这项试点拨款申请对这一缺陷进行表征将导致未来的研究， 对伤害性感受和肿瘤发生的影响。 为了解决这一假设，我们提出了单一的具体目标来评估不同的影响 Rac1对髓鞘雪旺细胞和非髓鞘雪旺细胞转录本的激活。作为第一次 Step，将WT SCs的转录与早期SC靶向的小鼠异常的SCs进行比较 Rac1的激活。其次，我们将扩大分析范围，以包括后来激活了rac1的小鼠，因为这些 动物似乎有形态和功能正常的干细胞。第三，我们将通过以下方式验证这些数据 分析特定的转录本和蛋白质以验证转录本的变化。 如果成功，这些研究将开始阐明rac1在调节细胞周期中的重要分子功能 体内雪旺细胞的分化、生理功能和肿瘤意义 了解和治疗疼痛，并了解SC肿瘤的发生，如在神经纤维瘤。

PROJECT ABSTRACT Schwann cells (SCs) are neural crest derived cells whose primary function is the support and protection of neurons in the peripheral nervous system. SCs can be divided into two subtypes: myelinating and non- myelinating. Myelinating SCs form 1:1 relationships with neurons and ensheath them in myelin to provide rapid conduction of nerve impulses, such as those required for transduction of proprioception and touch. In contrast, non-myelinating SCs typically bundle multiple axons into nerve structures known as Remak bundles, which support the cells but do not speed transmissions. Individuals with dysfunction of SCs exhibit a variety of phenotypes which may include decreased sensation, weakness, and pain, and these diseases are among the most common inherited conditions in humans. In addition, SCs are also the cell type targeted for tumorigenesis in the Neurofibromatosis syndromes (NF1 and NF2) which ae associated with benign and malignant tumors of the peripheral nerves and other locations. Rac1 is a small GTPase protein of the Rho family, and it has been shown to essential for tumorigenesis in both NF1- and NF2-associated tumors, as well as other forms of inherited Schwann cell tumors (e.g., Carney Complex). Recently, analyses of inherited SC dysfunctional syndromes have also shown dysregulation of Rac1. To investigate the role of Rac1 signaling in SC function and tumorigenesis, my laboratory has generated a mouse model carrying a conditionally active allele of Rac1 and performed preliminary characterization of the effects of activation Rac1 at various stages of SC development. We observe that activation of Rac1 in early embryogenesis enhanced cell proliferation and affects the ability of non- myelinating SCs to form normal Remak bundles, which are completely absent from nerves with Rac1 activated in early embryogenesis. In this R03 application, we hypothesize that Rac1 activation leads to aberrant differentiation specifically in non-myelinating SCs without affecting the trajectory of myelinating SCs. Initial characterization of this defect through this pilot grant application will lead to future studies with important implications for nociception and for tumorigenesis. To address this hypothesis, we propose the single Specific Aim to assess the differential effects of activation of Rac1 on the transcriptomes of myelinating and non-myelinating Schwann cells. As a first step, will compare the transcriptomes of WT SCs to the abnormal SCs in mice with SC-targeted early activation of Rac1. Secondly, we will expand the analysis to include mice with later activation of Rac1, as these animals appear to have morphologically and functional normal SCs. Thirdly, we will validate these data by analyzing specific transcripts and proteins to verify the transcriptomic changes. If successful, these studies will begin to elucidate the important molecular functions of Rac1 in mediating the differentiation, physiologic function, and tumor implications of Schwann cells in vivo, with implications both for understanding and treating pain, and for understanding SC tumorigenesis such as in NF.