Single cell transcriptomics of nerves that lack Remak bundles

项目摘要 雪旺细胞（SC）是神经嵴来源的细胞，其主要功能是支持和保护神经细胞。 周围神经系统中的神经元SC可分为两种亚型：髓鞘形成型和非髓鞘形成型。 髓鞘形成髓鞘化SC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 传导神经冲动，如本体感觉和触觉的传导所需的神经冲动。与此相反， 非髓鞘形成SC通常将多个轴突捆绑成称为Remak束的神经结构， 支持小区但不加速传输。具有SC功能障碍的个体表现出多种 表型，可能包括感觉减退，虚弱和疼痛，这些疾病是 人类最常见的遗传病此外，SC也是肿瘤发生的靶细胞类型 神经纤维瘤综合征（NF 1和NF 2）与良性和恶性肿瘤有关， 周围神经和其他部位。Rac 1是Rho家族的小GT3蛋白，并且它已经被 在NF 1和NF 2相关肿瘤以及其他形式的遗传性肿瘤中， 雪旺细胞肿瘤（例如，Carney Complex）。最近，遗传性SC功能障碍综合征的分析， 也显示了Rac 1的失调。为了研究Rac 1信号在SC功能和肿瘤发生中的作用， 我的实验室已经建立了一个携带Rac 1条件活性等位基因的小鼠模型， 在SC发展的各个阶段激活Rac 1的影响的初步表征。我们观察 Rac 1在早期胚胎发生中的激活增强了细胞增殖，并影响了非- 髓鞘化SC以形成正常的Remak束，其在Rac 1激活的神经中完全不存在 在早期胚胎发育中。在该R 03应用中，我们假设Rac 1激活导致异常的 在不影响髓鞘形成SC的轨迹的情况下，可以特异性地在非髓鞘形成SC中诱导分化。初始 通过这一试点赠款申请这一缺陷的表征将导致未来的研究与重要的 对伤害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，以评估不同的影响， 在髓鞘形成和非髓鞘形成的雪旺细胞的转录组上激活Rac 1。作为第一 第一步，将比较WT SC的转录组与具有SC靶向的早期肿瘤的小鼠中的异常SC。 激活Rac 1。其次，我们将扩大分析范围，包括Rac 1激活较晚的小鼠，因为这些小鼠 动物似乎具有形态和功能正常的SC。第三，我们将验证这些数据， 分析特定的转录物和蛋白质以验证转录组学变化。 如果成功的话，这些研究将开始阐明Rac 1在介导细胞凋亡中的重要分子功能。 体内雪旺细胞的分化，生理功能和肿瘤影响，以及对 了解和治疗疼痛，并了解SC肿瘤发生，如NF。

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.