Molecular Mechanisms Controlling Somatosensory Neuron Development

描述(申请人提供)：神经元与雪旺细胞的相互作用对周围神经系统(PNS)的发育、功能和再生非常重要。这种串扰会导致衰弱的疾病，如夏科-玛丽-图斯病和其他外周神经病。然而，轴突和不同类型的雪旺细胞之间相互作用的分子本质以及这些相互作用的调节还不完全清楚。在这项提案中，我将确定转录因子Er81如何控制环状小体的发育，环状小体是由单个有髓感觉轴突和非髓鞘小体形成的雪旺细胞组成的特殊感觉末梢器官。Er81突变小鼠没有环状小体，我的初步数据表明，主要缺陷来自于轴突-雪旺细胞相互作用的中断。由于Er81是一种转录因子，这种效应必须由其他下游效应器执行。神经调节蛋白-1(Nrg1)是轴突-雪旺细胞相互作用的关键调节因子。富含半胱氨酸胞外域的Nrg1亚型(Nrg1-CRD)的表达水平决定了外周轴突髓鞘形成的厚度。此外，包含感觉轴突、肌肉纤维和非髓鞘雪旺细胞的肌梭的发育需要其他Nrg1亚型，最有可能的是那些具有Ig样胞外域(Nrg1-Ig)的亚型。尽管Nrg1具有重要的功能，但Nrg1如何被调节以获得适当的表达水平仍是完全未知的。有趣的是，我发现Er81突变小鼠的躯体感觉神经元中Nrg1的两种异构体的表达减少，周围轴突的髓鞘厚度减少。因此，我假设Er81调节Nrg1的多种异构体的表达，以控制轴突与髓鞘和非髓鞘雪旺细胞的相互作用。在目标1中，我将确定ER81在神经元中是维持大直径体感轴突的高水平髓鞘形成所必需的。我将量化Er81突变体感神经元中Nrg1-CRD和Nrg1-Ig下调的程度，并证明Er81通过直接与Nrg1启动子结合来调节Nrg1的表达。为了将Nrg1放在Er81下游的环状小体发育中，我将通过异位供应Nrg1来挽救Er81突变小鼠的环状小体。在目标2中，我将通过消融来自体感神经元的所有Nrg1亚型来确定Nrg1在环岛小体形成中的作用。我还将通过抢救实验和分析同种异型突变小鼠来确定Nrg1的哪些亚型是小体形成所必需的。最后，我将检查高水平的Nrg1是否足以在一个异位的位置--小鼠足垫--诱导环状小体的形成。总而言之，我的工作预期的结果将确定FIRS转录机制，该机制通过调节Nrg1来控制PNS轴突的髓鞘形成程度，并阐明神经元与非髓鞘雪旺细胞相互作用的分子机制。

DESCRIPTION (provided by applicant): Neuron-Schwann cell interactions are important for the development, function, and regeneration of the peripheral nervous system (PNS). Perturbations in this crosstalk can cause debilitating diseases, such as Charcot- Marie-Tooth disease and other peripheral neuropathies. However, the molecular nature of the interactions between axons and different classes of Schwann cells and the regulation of these interactions are not fully understood. In this proposal, I will determine how the transcription factor Er81 controls development of Pacinian corpuscles, which are specialized sensory end organs composed of a single myelinated sensory axon and non-myelinating corpuscle-forming Schwann cells. Er81 mutant mice have no Pacinian corpuscles, and my preliminary data suggest that the primary deficit comes from disrupted axon-Schwann cell interactions. Since Er81 is a transcription factor, this effect must be executed by other downstream effectors. Neuregulin-1 (Nrg1) is a key mediator of axon-Schwann cell interactions. The expression level of an isoform of Nrg1 with cysteine rich extracellular domain (Nrg1-CRD) determines the thickness of myelination of peripheral axons. In addition, the development of muscle spindles, which contain sensory axons, muscle fibers, and non-myelinating Schwann cells, requires other Nrg1 isoforms, most likely those with an Ig-like extracellular domain (Nrg1-Ig). Despite the important functions of Nrg1, how Nrg1 is regulated to acquire an appropriate expression level is completely unknown. Interestingly, I found that Er81 mutant mice display decreased expression of both isoforms of Nrg1 in somatosensory neurons and reduced myelination thickness of peripheral axons. Therefore, I hypothesize that Er81 regulates the expression of multiple isoforms of Nrg1 to control axonal interactions with both myelinating and non-myelinating Schwann cells. In Aim 1, I will determine that Er81 is required in neurons to maintain a high level of myelination of large-diameter somatosensory axons. I will quantify the extent of the downregulation of Nrg1-CRD and Nrg1-Ig in Er81 mutant somatosensory neurons, and demonstrate that Er81 regulates Nrg1 expression by direct binding to the Nrg1 promoter. To place Nrg1 downstream of Er81 in Pacinian corpuscle development, I will rescue Pacinian corpuscles in Er81 mutant mice by ectopically supplying Nrg1. In Aim 2, I will determine the role of Nrg1 in Pacinian corpuscle formation by ablating all isoforms of Nrg1 from somatosensory neurons. I will also determine which isoforms of Nrg1 are required for corpuscle formation through rescue experiments and analyzing isoform specific mutant mice. Lastly, I will examine if a high level of Nrg1 is sufficient to induce the formation of Pacinian corpuscles in an ectopic location, the mouse foot pad. Collectively, anticipated results from my work will identify the firs transcriptional mechanism that controls the extent of myelination of PNS axons via the regulation of Nrg1, and elucidate molecular mechanisms of neuronal interaction with non-myelinating Schwann cells.