Mechanisms of axon guidance in laryngeal reinnervation following injury of the recurrent laryngeal nerve

项目摘要：每年有数万名患者发生喉返神经(RLN)损伤 在美国的一年。它会导致合并性神经再支配导致声带瘫痪和严重 发音困难。这会导致显著的患者发病率，因为声音是每个声音的唯一和不可分割的部分 人的身份，几乎影响到我们日常生活的方方面面，从与亲人交谈，到参与 或者只是简单地完成日常任务。发声功能障碍会导致恐惧和孤立，以及受损 生活质量、残疾索赔和工人生产力丧失，类似于严重的慢性病，导致 沉重的社会负担。目前的治疗方法是次优的，而且还没有可以恢复嗓音的治疗方法 折叠动作和正常的声音。我们对分子机制的认识存在着严重的空白 神经再生过程中潜在的轴突引导。我们的长期目标是识别表情和 引导线索的作用并操纵它们来引导选择性的、非联动的再神经支配 喉肌损伤后，声带功能恢复正常，嗓音正常。 我们先前的工作表明，在胚胎发育和喉返神经损伤后，喉部肌肉 是以相似的顺序被支配的。然而，喉返神经损伤后的再神经支配是紊乱的，导致联会运动。 有声带功能障碍。我们的实验室已经确定Netrin-1和GDNF是最有可能发挥作用的因子 RLN再生过程中轴突引导的中心作用。我们已经证明了它们在喉部的表达 肌肉及其受体在疑核(NA)的表达按时间顺序协调 有轴突再神经支配。我们已经证明，操纵这些指导线索可以改变 神经再支配的模式。 这些发现表明，引导线索的表达受到控制，但功能失调，导致了联动 喉返神经损伤后的再神经支配和声带瘫痪。相比之下，发育神经，也就是 也严格控制时间，导致正常的声带功能。我们的中心假设是 Netrin-1和GDNF是喉神经再支配的重要指导信号。此外，对他们的操纵 RLN损伤后模仿正常发育过程中所见的通路将导致选择性的、非 恢复正常声带功能的联合神经再支配。在强劲的初步数据指引下，我们 寻求追求以下三个具体目标：1)描述GDNF、Netrin-1、 以及它们的受体在喉发育的神经支配过程中的作用2)来描述调节 GDNF、Netrin-1及其受体在发育神经支配和再神经支配中的作用 RLN损伤后；3)体外评价NA和肌肉中信号通路的调节作用。这 研究将广泛影响神经再生领域，促进我们对神经再生机制的了解 轴突寻路有可能改变声带麻痹的治疗。

PROJECT SUMMARY: Recurrent laryngeal nerve (RLN) injury occurs in tens of thousands of patients per year in the United States. It results in synkinetic reinnervation inducing vocal fold paralysis and severe dysphonia. This produces significant patient morbidity as the voice is a unique and integral part of each person's identity, impacting virtually every aspect of our daily lives, from speaking to loved ones, to engaging in business or simply completing daily tasks. Voice dysfunction leads to fear and isolation, as well as impaired quality of life, disability claims and lost worker productivity similar to severe chronic diseases, resulting in a significant societal burden. Current therapies are suboptimal and there are no treatments that can restore vocal fold motion and normal voice. There are critical gaps in our knowledge about the molecular mechanisms underlying axonal guidance during nerve regeneration. Our long-term goals are to identify the expression and actions of guidance cues and to manipulate them to guide selective, non-synkinetic reinnervation of the laryngeal muscles after RLN injury, with restoration of normal vocal fold function and normal voice. Our previous work has revealed that in both embryologic development and post-RLN injury, laryngeal muscles are innervated in similar sequences. Yet, post-RLN injury reinnervation is disordered, resulting in synkinesis with vocal fold dysfunction. Our laboratory has identified Netrin-1 and GDNF as the factors most likely to play a central role in axon guidance during RLN regeneration. We have shown that their expression in laryngeal muscles and the expression of their receptors in the nucleus ambiguus (NA) are chronologically coordinated with axonal reinnervation. We have demonstrated that manipulation of these guidance cues can alter the pattern of reinnervation. These findings suggest a controlled but dysfunctional expression of guidance cues resulting in synkinetic reinnervation and vocal fold paralysis after RLN injury. In comparison, developmental innervation, which is also tightly controlled chronologically, results in normal vocal fold function. Our central hypothesis is that Netrin-1 and GDNF are critical guidance cues in laryngeal reinnervation. Further, manipulation of their pathways post-RLN injury to mimic those seen during normal development will in result in selective, non- synkinetic reinnervation with restoration of normal vocal fold function. Guided by strong preliminary data, we seek to pursue the following three Specific Aims: 1) to describe the patterns of expression of GDNF, Netrin-1, and their receptors during innervation of the larynx in development 2) to describe the pathways that modulate the function of GDNF, Netrin-1 and their receptors during innervation in development and during reinnervation post-RLN injury and 3) to evaluate in vitro the effects of modulating pathways in the NA and muscle. This research will broadly impact the field of nerve regeneration, advancing our knowledge of the mechanisms of axonal pathfinding with the potential to transform the treatment of vocal fold paralysis.