Molecular Mechanisms Controlling Somatosensory Neuron Development

描述（由申请人提供）：神经元-许旺细胞相互作用对于周围神经系统（PNS）的发育、功能和再生非常重要。这种串扰中的扰动可引起使人衰弱的疾病，例如夏科-玛丽-图思病和其他周围神经病。然而，轴突和不同类别的许旺细胞之间的相互作用的分子性质和这些相互作用的调节还没有完全理解。在这个提议中，我将确定转录因子Er81如何控制Pacinian小体的发育，Pacinian小体是由单个有髓鞘的感觉轴突和非髓鞘小体形成的Schwann细胞组成的专门的感觉末端器官。Er81突变小鼠没有Pacinian小体，我的初步数据表明，主要缺陷来自轴突-许旺细胞相互作用的破坏。由于Er81是一个转录因子，这种作用必须由其他下游效应子来执行。神经调节蛋白-1（Nrg1）是轴突-许旺细胞相互作用的关键介质。具有富含半胱氨酸的胞外结构域（Nrg1-CRD）的Nrg1亚型的表达水平决定外周轴突髓鞘形成的厚度。此外，包含感觉轴突、肌纤维和非髓鞘化雪旺细胞的肌梭的发育需要其他Nrg 1亚型，最有可能是那些具有Ig样胞外结构域（Nrg 1-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是完全未知的。有趣的是，我发现Er81突变小鼠显示减少的表达的两种异构体的Nrg1的躯体感觉神经元和减少髓鞘厚度的外周轴突。因此，我假设Er81调节多种亚型的Nrg1的表达，以控制轴突与髓鞘形成和非髓鞘形成雪旺细胞的相互作用。在目标1中，我将确定Er81是神经元维持高水平的大直径体感轴突髓鞘形成所必需的。我将量化的程度下调的Nrg1-CRD和Nrg1-IG在Er81突变体体感神经元，并证明Er81调节Nrg1的表达，直接结合到Nrg1启动子。为了将Nrg 1置于Er81的环层小体发育的下游，我将通过异位供应Nrg 1来拯救Er81突变小鼠的环层小体。在目的2中，我将通过从体感神经元中去除Nrg 1的所有亚型来确定Nrg 1在环层小体形成中的作用。我还将通过拯救实验和分析亚型特异性突变小鼠来确定Nrg1的哪些亚型是小体形成所需的。最后，我将检查高水平的Nrg 1是否足以诱导在异位位置（小鼠足垫）形成环层小体。总的来说，我的工作的预期结果将确定第一个转录机制，通过Nrg1的调节控制PNS轴突的髓鞘形成的程度，并阐明神经元与非髓鞘形成的许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.