Neuronal P-Rex1 repression: a key factor in early-life environmental cigarette smoke exposure mediated risk of asthma

早期接触环境香烟烟雾(ECS)改变呼吸道神经并增加发病率 哮喘在晚年的发病率很高，但其机制仍不明确。我们最近发现神经元P-REx1是一种 小鼠呼吸道神经支配的重要调节因子及其表达明显下调 早期生命的ECS。目的：明确P-REx1抑制在早期ECS中的机制和重要性。 诱导的呼吸道高神经和高反应性(AHR)，哮喘的病理生理标志。 长期目标：确定靶向神经元P-REx1是否为预防 早期生活中与ECS相关的哮喘进展。研究结果：1)P-REx1在神经元中高表达，而在呼吸道中不表达 细胞。2)P-REx1基因敲除(KO)小鼠表现为气道平滑肌(ASM)神经支配过度和AHR。WT小鼠 暴露于早期生命的ECS表现出相似的表型，迷走神经节中P-REx1的表达减少了60%。割断 迷走神经使这些小鼠的AHR减弱。3)ECS暴露可增强脑源性神经营养因子 ASM细胞分泌脑源性神经营养因子可作为小鼠迷走神经突起生长的靶源性信号 体外培养的神经元。4)P-Rex1过表达可阻断BDNF刺激的轴突生长，而P-Rex1表达缺失 使这些神经元对脑源性神经营养因子刺激明显增敏。5)内皮细胞升高的白介素6下调P- REx1，并增强BDNF刺激的轴突生长，该生长可被PKC抑制剂阻断。假设：IL-6 神经元P-REx1的抑制在早期ECS诱导的ASM过度神经支配和AHR中起关键作用 哮喘的症状。我们将使用分子、细胞和动物模型来验证这一假设。目标1：阐明 早期ECS暴露诱导神经元P-REx1抑制的机制。我们假设IL-6 通过PKC依赖机制抑制神经元P-REx1。我们将首先使用siRNA来沉默P-REx1在 小鼠迷走感觉神经元P-Rex1在脑源性神经营养因子增强IL-6中的作用 轴突生长。然后，我们将研究P-REx1表达的恢复是否减弱了IL-6的刺激效应。 最后，我们将使用抑制剂和siRNAs来确定与IL-6诱导的P-REx1有关的PKC亚型 抑制和轴突生长。目的2：探讨神经元型P-Rex1的病理意义 与早期ECS暴露相关的哮喘的抑制。我们假设IL-6对神经元P-REx1的抑制 是早期ECS相关哮喘发生和严重程度的关键决定因素。WT和P-REx1 KO 从出生后第2天(PND)开始，小鼠将暴露在ECS或空气中10天。 小鼠再次接触ECS或过敏原屋尘螨后24小时的有创性气管切开术 PND59.早期ECS暴露对ASM神经支配和表型(重塑、收缩)的影响 接受检查。P-REx1缺失是否加重早期ECS诱导的ASM过度神经支配和AHR 将会被确定。最后，我们将确定口服IL-6抑制剂LMT-28是否会改善 早期ECS通过阻止P-REx1抑制和ASM过度神经支配诱导小鼠AHR。

Early-life environmental cigarette smoke (ECS) exposure alters airway innervation and increases the incidence of asthma later in life, but the mechanisms remain undefined. We recently identified neuronal P-Rex1 as an important regulator of airway innervation and its expression was markedly down-regulated in mice exposed to early-life ECS. Objective: To define the mechanism and importance of P-Rex1 repression in early-life ECS- induced airway hyperinnervation and hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Long-term goal: to determine whether targeting neuronal P-Rex1 provides a new strategy for preventing early-life ECS-related asthma progression. Findings: 1) P-Rex1 is highly expressed in neurons but not airway cells. 2) P-Rex1 knockout (KO) mice exhibit airway smooth muscle (ASM) hyperinnervation and AHR. WT mice exposed to early-life ECS showed similar phenotypes with 60% reduction of P-Rex1 in vagal ganglia. Severing vagus nerves attenuated AHR of these mice. 3) ECS exposure enhances brain-derived neurotrophic factor (BDNF) secretion from ASM cells, serving as a target-derived signal for neurite growth of mouse vagal sensory neurons in vitro. 4) P-Rex1 over-expression blocked BDNF-stimulated neurite growth whereas loss of P-Rex1 markedly sensitized these neurons to BDNF stimulation. 5) ECS-elevated interleukin (IL)-6 down-regulates P- Rex1 and enhances BDNF-stimulated neurite growth that is blocked by a PKC inhibitor. Hypothesis: IL-6 repression of neuronal P-Rex1 plays a crucial role in early-life ECS-induced ASM hyperinnervation and AHR of asthma. We will test this hypothesis using molecular, cellular, and animal models. Aim 1: To elucidate the mechanism of early-life ECS-exposure-induced neuronal P-Rex1 repression. We hypothesize that IL-6 represses neuronal P-Rex1 via a PKC-dependent mechanism. We will first use siRNAs to silence P-Rex1 in mouse vagal sensory neurons to assess the importance of P-Rex1 in IL-6 potentiation of BDNF-induced neurite growth. We will then investigate if restoration of P-Rex1 expression attenuates IL-6 stimulatory effects. Finally, we will use inhibitors and siRNAs to identify the PKC isoforms responsible for IL-6-induced P-Rex1 repression and neurite growth. Aim 2: To investigate the pathologic importance of neuronal P-Rex1 repression in early-life ECS exposure-related asthma. We hypothesize that IL-6 repression of neuronal P-Rex1 is a critical determinant in the development and severity of early-life ECS-related asthma. WT and P-Rex1 KO mice will be exposed to ECS or air for 10 days beginning on postnatal day (PND) 2. AHR will be assessed by invasive tracheostomy 24h after a re-exposure of mice to acute insult of ECS or allergen house dust mite on PND59. Effects of early-life ECS exposure on ASM innervation and phenotype (remodeling, contractility) will be examined. Whether loss of P-Rex1 exacerbates early-life ECS-induced ASM hyper-innervation and AHR will be determined. Finally, we will determine whether oral administration of IL-6 inhibitor LMT-28 ameliorates early-life ECS-induced mouse AHR by preventing P-Rex1 repression and ASM hyperinnervation.