In vivo real-time monitoring of reactive oxygen species and opioid signaling in a model for opioid receptor activity.

摘要 2017年，美国卫生与公众服务部宣布正在进行的阿片类药物流行是公开的 在那一年，超过4.7万名美国人死于阿片类药物过量，引发了健康危机。一个关键的部分， 解决办法是了解大脑回路对阿片类药物刺激的基本反应和适应。为 例如，阿片受体的脱敏是疼痛管理中的一个关键问题，因为它需要 增加止痛化合物的剂量，这可能会导致药物成瘾。最近，它 已经证明，神经元中的u和kappa阿片受体的激活引起反应性的产生。 氧物种(ROS)通过一条涉及NADPH氧化酶和c-jun氨基末端激酶的途径。因此， 这种位于受体下游的独特反应可以用来检测特定的阿片受体激活。 和调制。目前阿片受体的研究要么依赖于细胞培养的体外实验，要么依赖于分析 以监测他们在药物暴露下的情况。我们目前缺乏灵敏的荧光传感器， 这将使我们能够利用最先进的光纤光度法直接监测mU-阿片受体(MOR) 实时和活体活动。当代传感器目前的局限性是响应时间慢、低 特异性、低信号输出、毒性或对体外组织制备的依赖性。我们的目标是开发一种 基因编码的传感器蛋白，检测内源水平的ROS水平和响应时间 以及能够在体内监测MOR激活时的神经系统的信号幅度。我们 最近开发了一种新型的荧光ROS传感器，方法是将绿色荧光蛋白融合到细菌中 过氧化氢结合蛋白。信号动力学、ROS敏感度和信号幅度显著 与其他可用的工具相比得到了增强。我们假设我们可以进一步提高这个工具的保真度 通过在过氧化氢结合位点和相互之间的界面上进行额外的结构引导蛋白质设计 绿色荧光报告器和感应域。我们的目标是在体内更多地表达这种新工具 并将ROS信号与MOR的药理活性联系起来。我们假设ROS发出信号 在MOR中，神经元在药物暴露下会增加。第二，我们假设我们将观察到 ROS在反复药物暴露下的短暂变化反映了MORS的脱敏。在这个项目的最后， 我们将拥有一种新型的、高度特异的传感器，用于监测阿片受体的活性和适应性。我们的 这项提议意义重大，因为我们将首次能够在临床上监测这一技术的适应情况 体内阿片类药物暴露的相关信号通路。我们的方法是创新的，因为我们结合了 结构引导的蛋白质工程和体内监测阿片类药物触发的信号来解剖一个难以 进入神经元信号通路。此外，这种方法可以在未来的研究中广泛应用于 监测药物暴露期间阿片受体的活动水平，并将随后神经元的变化联系起来 刺激行为的信号和可塑性，或止痛耐受性。

ABSTRACT In 2017 the U.S. Department of Health and Human Services declared the ongoing opioid epidemic a public health crisis after more than 47,000 Americans died from opioid overdoses during that year. A critical part of the solution is to understand the fundamental reaction and adaptation of brain circuits to stimulation by opioids. For example, the desensitization of opioid receptors is a critical problem in pain management because it requires increasing doses of analgesic compounds, which could contribute to developing a drug addiction. Recently, it has been shown that the activation of mu and kappa opioid receptors in neurons cause the production of reactive oxygen species (ROS) through a pathway involving NADPH oxidase and c-Jun N-terminal kinase. Therefore, this distinct response, downstream from the receptor, could be utilized to detect specific opioid receptor activation and modulation. Current studies of opioid receptors rely either on in vitro experiments in cell cultures or analysis of ex-vivo brain tissue to monitor them under drug exposure. We currently lack sensitive fluorescent sensors, which would allow us to utilize state-of-the-art fiber photometry to directly monitor mu-opioid receptor (MOR) activity in real-time and in vivo. Current limitations of contemporary sensors are slow response times, low specificity, low signal output, toxicity, or dependency on ex vivo tissue preparation. Our goal is to develop a genetically encoded sensor protein that detects ROS levels at endogenous levels with response time and signal amplitudes that will enable in vivo monitoring of neuronal systems upon MOR activation. We have recently developed a novel fluorescent ROS sensor by fusing a green fluorescent protein to a bacterial hydrogen peroxide binding protein. Signal kinetics, ROS sensitivity, and signal amplitudes are significantly enhanced compared to other available tools. We hypothesize that we can further increase the fidelity of this tool by additional structure-guided protein design at the hydrogen-peroxide binding site and the interface between the green fluorescent reporter and the sensing domain. Our objective is to express this novel tool in vivo in MOR positive neurons and to link ROS signals to MOR activity pharmacologically. We hypothesize that ROS signals in MOR neurons will increase under drug exposure. Second, we hypothesize that we will observe a decrease of ROS transients under repeated drug exposure reflecting the desensitization of MORs. At the end of this project, we will have a novel and highly specific sensor for monitoring opioid receptor activity and adaptivity. Our proposal is significant because, for the first time, we will be able to monitor the adaptation of this clinically relevant signaling pathway to opioid exposure in vivo. Our approach is innovative because we combine structure-guided protein engineering and in vivo monitoring of opioid-triggered signals to dissect a difficult-to- access neuronal signaling pathway. Furthermore, this approach could be broadly applied in future studies to monitor the activity levels of opioid receptors during drug exposure and link the subsequent changes in neuronal signaling and plasticity to motivated behaviors, or analgesic tolerance.