Development of PNEC innervation and neuroplasticity after early life insult

摘要 肺神经内分泌细胞 (PNEC) 如何受神经支配以及神经在多大程度上调节 PNEC 发育和疾病中的功能是该领域的开放和基本问题。此举的目的 提案旨在解决这些基本问题并评估神经营养素 4 (NT4) 在这些问题中的关键作用 事件。使用 NT4-/- 小鼠系和过敏原暴露的新生小鼠模型，我们生成了 初步数据得出本提案将研究的 3 个假设： 1) PNEC 需要 NT4 发育过程中的神经支配以及早期生命损伤后 PNEC 神经支配的增加； 2）早 接触生活过敏原会改变支配 PNEC 的感觉传入神经和传出神经的功能 从而导致γ-氨基丁酸（GABA）分泌失调和长期杯状细胞化生； 3） 肺肥大细胞是生命早期接触过敏原后 NT4 水平升高的候选来源。至 综上所述，我们发现 NT4 在出生后发育过程中由 PNEC 表达，并充当营养因子 支配神经建立联系的因素。出生后肺部发育中的过敏原暴露 NT4 水平异常升高。在这种病理条件下，我们发现 PNEC 神经支配 增加与杯状细胞化生延长有关。值得注意的是，PNEC 是唯一的细胞来源。 肺部中的 GABA，是小鼠模型中过敏原诱导的杯状细胞化生所必需的信号以及相关的 人类哮喘患者和吸烟者的粘液分泌过多。此外，PNEC 需要 NT4 神经支配过度和 GABA 分泌失调，与其他研究中已建立的范式一致 神经内分泌系统即神经调节内分泌的分泌。这些初步发现表明 NT4 在发育过程中的 PNEC 神经支配和早期生命损伤后的神经可塑性中发挥重要作用， 通过比较 PNEC 神经支配的模式和程度，可以广泛表征 野生型和 NT4-/- 小鼠有或没有暴露于过敏原，使用不同类型神经的标记（目标 1）。将 NT4 诱导的 PNEC 过度神经支配与生命早期杯状细胞化生延长联系起来 过敏原暴露，目标 2 中提出的实验将评估感觉传入和 诱导 PNEC 分泌 GABA 的传出信号及其与 NT4 的关系。最后，鉴于 NT4 在病理条件下异常 PNEC 神经支配中的核心作用，我们检查了 NT4 表达 在受伤的肺部。我们发现在生命早期过敏原期间，肥大细胞群扩大、活化，表达 NT4 曝光。肺肥大细胞是否通过产生 NT4 来促进 PNEC 过度神经支配？ 评估（目标 3）。为了进一步增强疾病相关性，我们将验证小鼠研究的主要发现 婴儿灵长类动物损伤模型和人类肺部样本。该提案共同研究了复杂的 神经、PNEC 和出生后发育和损伤过程中炎症之间的相互作用。我们的发现 表明慢性气道疾病（例如哮喘）的发病机制可能涉及紊乱 早期侮辱之后的发育过程。我们期望完成拟议的 研究将提供有关肺神经内分泌系统如何形成和 功能。识别粘液过度产生的沿神经-PNEC轴的机制 可能为发现新的治疗策略奠定基础。

ABSTRACT How pulmonary neuroendocrine cells (PNECs) become innervated and to what extent nerves regulate PNEC function in development and diseases are open and basic questions for the field. The objective of this proposal is to address these fundamental issues and to assess the key role of neurotrophin 4 (NT4) in these events. Using a NT4-/- mouse line and a neonatal mouse model of allergen exposure, we generated preliminary data leading to 3 hypotheses that will be studied in this proposal: 1) NT4 is required for PNEC innervation during development and for the increases in PNEC innervation following early life insults; 2) early life allergen exposure alters the function of sensory afferents and efferent nerves that innervate PNECs thereby causing deregulated γ-aminobutyric acid (GABA) secretion and long-term goblet cell metaplasia; 3) pulmonary mast cells are a candidate source of elevated NT4 levels following early life allergen exposure. To summarize, we found that NT4 was expressed by PNECs during postnatal development and acted as a trophic factor for the innervating nerves to establish connection. Allergen exposure to developing, postnatal lungs aberrantly elevated the levels of NT4. Under this pathological condition, we discovered that PNEC innervation was increased associated with prolonged goblet cell metaplasia. Notably, PNECs were the only cell source of GABA in lungs, a signal essential for allergen-induced goblet cell metaplasia in mouse models and associated with mucous overproduction in human asthmatics and smokers. In addition, NT4 was required for PNEC hyperinnervation and deregulated GABA secretion, consistent with the established paradigm in other neuroendocrine systems that nerves regulate endocrine secretion. These preliminary findings point to an essential role for NT4 in PNEC innervation during development and neuroplasticity following early life injury, which will be extensively characterized by comparing the pattern and degree of PNEC innervation between wild type and NT4-/- mice with and without allergen exposure using markers for different types of nerves (Aim 1). To connect NT4-induced PNEC hyperinnervation to prolonged goblet cell metaplasia following early life allergen exposure, proposed experiments in Aim 2 will assess functional changes in sensory afferents and efferent signals that induce GABA secretion from PNECs and their relationships to NT4. Lastly, given the central role of NT4 in aberrant PNEC innervation under pathological conditions, we examined NT4 expression in injured lungs. We found an enlarged, activated mast cell population expresses NT4 during early life allergen exposure. Whether pulmonary mast cells contribute to PNEC hyperinnervation by producing NT4 will be evaluated (Aim 3). To further enhance disease relevance, we will validate key findings from the mouse work in infant primate models of injury and human lung samples. Together, this proposal investigates complex interactions between nerves, PNECs, and inflammation during postnatal development and injury. Our findings indicate that the pathogenesis of chronic airway diseases, such as asthma, may involve disrupted developmental processes following early episodes of insults. We expect that completion of the proposed studies will provide fundamental knowledge about how the pulmonary neuroendocrine system forms and functions. Identification of the mechanisms along the nerve-PNEC axis underlying mucous overproduction may lay the foundation for the discovery of new treatment strategies.