Metabotropic contributions to pH-sensitivity and breathing modulation by RTN chemoreceptors

项目概要/摘要 位于梯形后核 (RTN) 的一组离散神经元，表达转录因子， Phox2b 和神经肽 Neuromedin B (Nmb) 提供关键的兴奋驱动来调节下游 呼吸节律/模式生成电路。这些神经元的活动受到 CO2 变化的调节 （或 H ）以及各种其他感觉和唤醒状态输入。该神经系统的功能障碍与 潜在致命的综合症（例如婴儿猝死，SIDS；先天性中枢性通气不足，CCHS）和再治疗 CO2 阈值/敏感性的设置可能会伴随并加剧各种慢性呼吸疾病（例如， 慢性阻塞性肺病（COPD）。我们小组之前的工作已经确定了两个 CO2/H 传感器 RTN 神经元中：质子激活的 GPCR、GPR4 和质子失活的钾通道 TASK-2。 大多数 RTN 神经元表达这两种传感器，但尚不清楚这两种蛋白是否提供冗余或 是细胞对 H 浓度增加的不同反应的基础。在这方面，RTN 神经元是高度 富含神经肽 PACAP 的表达，该神经肽与 SIDS 有关，我们的实验室 已显示有助于 CO2 调节呼吸（呼吸化学反射）：从 RTN 中删除 PACAP 神经元减弱呼吸化学反射，将 PACAP 注射到 RTN 靶向呼吸核中可增强呼吸化学反射 呼吸输出量。代谢信号，例如由 GPR4 激活引发的信号，被认为发挥着关键作用。 与突触释放小递质相比，致密核心囊泡释放神经肽的作用 囊泡。因此，我假设 GPR4 介导的 pH 敏感性和 cAMP 升高对于 RTN 神经元释放 PACAP 来控制中枢化学反射的各个方面。具体来说 目标1，我使用转基因小鼠来测试GPR4本身是否需要pH敏感性 细胞和生理作用，并利用脑干中的电生理学来检查下游的作用 RTN 神经元 pH 敏感性中的 Gαs 耦合信号传导，特别是腺苷酸环化酶激活。我的初步 CRISPR 修饰的新型敲入小鼠系和 cAMP 的药理学操作的数据是 与这个假设一致。在具体目标 2 中，我使用病毒方法来表达基因编码的、 光激活腺苷酸环化酶（bPAC）来测试这种特殊形式的代谢信号传导是否赋予 释放兴奋性神经肽 PACAP 的能力，支持 RTN 二氧化碳刺激的呼吸 神经元。我已经准备了一种用于 RTN 选择性表达 bPAC 的病毒，并实现了基于细胞的光学 体外检测 RTN 神经元 PACAP 释放的系统。总的来说，拟议的研究将提供新颖的 有关调节 RTN pH 敏感性和下游作用的分子机制的信息 呼吸调节过程中的神经元，更一般地说，cAMP 介导的信号在神经肽中的作用 释放。这些新分子机制的鉴定可能为疾病提供新的治疗靶点 呼吸。

PROJECT SUMMARY/ABSTRACT A discrete group of neurons located in the retrotrapezoid nucleus (RTN) that express the transcription factor, Phox2b and the neuropeptide, Neuromedin B (Nmb) provide a crucial excitatory drive to regulate downstream respiratory rhythm/pattern-generating circuits. The activity of these neurons is modulated by changes in CO2 (or H+) and various other sensory and arousal-state inputs. Dysfunction of this neuronal system is implicated in potentially fatal syndromes (e.g., sudden infant death, SIDS; congenital central hypoventilation, CCHS) and re- setting of CO2 threshold/sensitivity can accompany and exacerbate various chronic disorders of breathing (e.g., chronic obstructive pulmonary disease, COPD). Previous work in our group has identified two CO2/H+ sensors in RTN neurons: the proton-activated GPCR, GPR4 and the proton-inactivated potassium channel, TASK-2. Most RTN neurons express both sensors but it is unclear whether the two proteins provide redundancy or underlie different cellular responses to increased H+ concentration. In this respect, RTN neurons are highly enriched in expression of a neuropeptide, PACAP, that has been implicated in SIDS and which our laboratory has shown contributes to CO2-regulated breathing (respiratory chemoreflex): deletion of PACAP from RTN neurons blunts the respiratory chemoreflex, and PACAP injection into RTN-targeted respiratory nuclei enhances respiratory output. Metabotropic signaling, such as that initiated by GPR4 activation, is thought to play a critical role in neuropeptide release from dense core vesicles compared to small transmitter release from synaptic vesicles. Thus, I hypothesize that GPR4-mediated pH-sensitivity and cAMP elevation is crucial for the release of PACAP from RTN neurons to control aspects of the central chemoreflex. In Specific Aim 1, I use genetically modified mice to test whether the pH sensitivity of GPR4, per se, is required for its cellular and physiological actions, and use electrophysiology in brainstem to examine the role of downstream Gαs-coupled signaling, specifically adenylyl cyclase activation, in pH sensitivity of RTN neurons. My preliminary data with a novel line of CRISPR-modified knock-in mice and pharmacological manipulation of cAMP are consistent with this hypothesis. In Specific Aim 2, I use a viral approach to express a genetically-encoded, photo-activated adenylyl cyclase (bPAC) to test whether this particular form of metabotropic signaling confers the ability to release an excitatory neuropeptide, PACAP, that supports CO2-stimulated breathing by RTN neurons. I have prepared a virus for RTN-selective expression of bPAC, and implemented a cell-based optical system to detect PACAP release from RTN neurons in vitro. Collectively, the proposed studies will provide novel information regarding molecular mechanisms that regulate the pH sensitivity and downstream actions of RTN neurons during breathing regulation and, more generally, the role of cAMP-mediated signaling in neuropeptide release. Identification of these novel molecular mechanisms may provide new therapeutic targets for disorders of breathing.