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

项目概要/摘要 位于后斜方核（RTN）中表达转录因子的一组离散神经元， Phox 2 B和神经肽神经介肽B（Nmb）提供了一种重要的兴奋性驱动，以调节下游 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 (or H+）和其他各种感觉和唤醒状态输入。这种神经系统的功能障碍与以下因素有关： 潜在的致命综合征（例如，婴儿猝死，SIDS;先天性中枢性通气不足，CCHS）和再 CO2阈值/灵敏度的设定可伴随并加重各种慢性呼吸障碍（例如， 慢性阻塞性肺疾病（COPD）。我们小组之前的工作已经确定了两个CO2/H+传感器 在RTN神经元中：质子激活的GPCR，GPR 4和质子失活的钾通道，ASK-2。 大多数RTN神经元表达这两种传感器，但目前还不清楚这两种蛋白质是否提供了冗余， H+浓度增加时细胞的不同反应。在这方面，RTN神经元高度 富含神经肽PACAP的表达，PACAP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 神经元钝化呼吸化学反射，PACAP注射到RTN靶向的呼吸核中， 呼吸输出量促代谢信号传导，例如由GPR 4激活启动的信号传导，被认为在代谢过程中起关键作用。 与突触释放的小递质相比，神经肽从致密核心囊泡释放的作用 囊泡因此，我推测GPR 4介导的pH敏感性和cAMP升高对于 从RTN神经元释放PACAP以控制中枢化学反射的各个方面。在特定 目的1，我使用转基因小鼠来测试GPR 4本身的pH敏感性是否是其生长所必需的。 细胞和生理作用，并使用脑干电生理检查下游的作用， RTN神经元pH敏感性中的Gα s偶联信号，特别是腺苷酸环化酶激活。我的初步 一种新的CRISPR修饰的基因敲入小鼠品系和cAMP的药理学操作的数据是 与这一假设相一致。在《特定目标2》中，我用病毒的方法来表达一种基因编码的， 光活化腺苷酸环化酶（bPAC），以测试这种特殊形式的代谢信号是否赋予 释放兴奋性神经肽PACAP的能力，通过RTN支持CO2刺激呼吸 神经元我已经制备了一种用于RTN选择性表达bPAC的病毒，并实施了一种基于细胞的光学显微镜。 系统检测RTN神经元释放PACAP。总的来说，拟议的研究将提供新的 关于调节RTN的pH敏感性和下游作用的分子机制的信息 神经元在呼吸调节过程中的作用，更普遍的是，cAMP介导的信号传导在神经肽 release.这些新的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.