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

早期暴露于环境香烟烟雾（ECS）改变了气道神经支配， 哮喘在以后的生活中，但机制仍然不明确。我们最近发现神经元P-Rex 1是一种 呼吸道神经支配的重要调节因子，其表达在暴露于 早期的ECS目的：探讨P-Rex 1抑制在早期内皮细胞系统中的作用机制。 诱导的气道高神经支配和高反应性（AHR），哮喘的病理生理标志。 长期目标：确定靶向神经元P-Rex 1是否提供了一种新的预防策略。 早期ECS相关哮喘进展。结果：1）P-Rex 1在神经元中高表达，但在气道中不表达 细胞2)P-Rex 1基因敲除（KO）小鼠表现出气道平滑肌（ASM）神经支配过度和AHR。WT小鼠 暴露于早期ECS的小鼠表现出相似的表型，迷走神经节中P-Rex 1减少60%。切断 迷走神经可减弱AHR。3)ECS暴露增强脑源性神经营养因子 ASM细胞分泌的BDNF，作为小鼠迷走神经感觉神经突起生长的靶源性信号。 体外培养的神经元。4)P-Rex 1的过表达阻断了BDNF刺激的神经突起生长，而P-Rex 1的缺失则抑制了BDNF刺激的神经突起生长。 这些神经元对BDNF刺激明显敏感。5)ECS升高的白细胞介素（IL）-6下调P- Rex 1和增强BDNF刺激的神经突生长，其被PKC抑制剂阻断。假设：IL-6 神经元P-Rex 1的抑制在早期生命ECS诱导的ASM神经支配过度和AHR中发挥关键作用 哮喘我们将使用分子、细胞和动物模型来检验这一假设。目的1：阐明 早期生命ECS暴露诱导的神经元P-Rex 1抑制的机制。我们假设IL-6 通过PKC依赖性机制抑制神经元P-Rex 1。我们将首先使用siRNA沉默P-Rex 1， 小鼠迷走神经感觉神经元，以评估P-Rex 1在IL-6增强BDNF诱导的 神经突生长然后我们将研究P-Rex 1表达的恢复是否减弱IL-6的刺激作用。 最后，我们将使用抑制剂和siRNA来鉴定负责IL-6诱导的P-Rex 1的PKC亚型。 抑制和神经突生长。目的2：探讨神经元P-Rex 1的病理意义 抑制早期生命ECS呼吸相关哮喘。我们假设IL-6抑制神经元P-Rex 1 是早期ECS相关哮喘发展和严重程度的关键决定因素。WT和P-Rex 1 KO 从出生后第2天（PND）开始，将小鼠暴露于ECS或空气中10天。AHR将通过以下方式进行评估： 在小鼠再次暴露于ECS或过敏原屋尘螨急性损伤后24小时， PND 59。早期ECS暴露对ASM神经支配和表型（重塑，收缩性）的影响将 接受检查。P-Rex 1的缺失是否加重早期生命ECS诱导的ASM神经支配过度和AHR 将被确定。最后，我们将确定口服IL-6抑制剂LMT-28是否能改善 通过阻止P-Rex 1抑制和ASM神经支配过度，早期生命ECS诱导的小鼠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.