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

项目摘要/摘要 位于梯形后核(RTN)的一组离散的神经元，表达转录因子， PHOX2B和神经肽Neuromedin B(NMB)为调节下游提供了关键的兴奋性驱动 呼吸节律/模式产生电路。这些神经元的活动受到二氧化碳变化的调节。 (或H)和其他各种感觉和唤醒状态输入。这个神经系统的功能障碍牵涉到 潜在的致命综合征(例如，婴儿猝死，SID；先天性中枢性换气不足，CCHS)和Re 二氧化碳阈值/敏感度的设置会伴随和加重各种慢性呼吸紊乱(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二氧化碳/氢传感器 在RTN神经元中：质子激活的GPCRGPR4和质子失活的钾通道TASK-2。 大多数RTN神经元都表达这两种感受器，但目前尚不清楚这两种蛋白是提供冗余还是 这是不同细胞对H浓度增加的反应的基础。在这方面，RTN神经元是高度 富含一种神经肽PACAP的表达，该神经肽与小岛屿发展中国家有关，我们的实验室 已显示有助于二氧化碳调节的呼吸(呼吸化学反射)：从RTN中删除PACAP 神经元钝化呼吸化学反射，将PACAP注射到RTN靶向呼吸核团增强 呼吸量。新陈代谢信号，例如由GPR4激活启动的信号，被认为起着关键的作用 致密核心小泡释放神经肽与突触释放小递质的比较 水泡。因此，我推测GPR4介导的pH敏感性和cAMP升高对 从RTN神经元释放PACAP来控制中枢化学反射的各个方面。具体而言 目的1，我使用转基因小鼠来测试GPR4本身对pH的敏感性是否是其 细胞和生理活动，并利用脑干的电生理学来检查下游的作用 GαS偶联的信号转导，特别是腺苷环化酶的激活，在延髓核神经元的pH敏感性中起作用。我的初选 一种新的CRISPR修饰的敲入小鼠和cAMP的药理操作的数据是 与这一假设一致。在特定的目标2中，我使用病毒方法表达了一种基因编码的， 光激活的腺酰环化酶(BPAC)来测试这种特殊形式的代谢性信号是否 释放兴奋性神经肽PACAP的能力，该神经肽支持RTN刺激的二氧化碳呼吸 神经元。我已经准备了一种针对RTN选择性表达BPAC的病毒，并实现了基于细胞的光学 检测体外培养的RTN神经元释放PACAP的方法。总的来说，拟议的研究将提供新的 调节pH敏感性和RTN下游作用的分子机制的信息 呼吸调节中的神经元，更广泛地说，cAMP介导的信号在神经肽中的作用 放手。识别这些新的分子机制可能会为疾病的治疗提供新的靶点 呼吸的感觉。

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.