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

阿片受体活性模型中活性氧和阿片信号传导的体内实时监测。

• 批准号: 10369709
• 负责人: Andre Berndt
• 金额: $ 20.83万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10369709
• 关键词: Addictive Behavior; American; Analgesics; Animals; Anxiety; Behavior; Binding Proteins; Binding Sites; Brain; Cell Culture Techniques; Cells; Coupling; Dependence; Detection; Dose; Drug Addiction; Drug Exposure; Drug Prescriptions; Drug Tolerance; Drug Withdrawal Symptoms; Emotions; Engineering; Enhancers; Enterobacteria phage P1 Cre recombinase; Epidemic; Equipment; Fentanyl; Fiber; Funding; Future; G Protein-Coupled Receptor Signaling; Goals; Green Fluorescent Proteins; Habenula; Health; Hydrogen Peroxide; In Vitro; Kinetics; Lead; Link; MAPK8 gene; Measurement; Measures; Medial; Modeling; Molecular; Monitor; Morphine; Mus; NADPH Oxidase; Names; National Institute of Drug Abuse; Neurobiology; Neuronal Plasticity; Neurons; Opiate Addiction; Opioid; Opioid Receptor; Opioid agonist; Output; Overdose; Pain; Pain management; Pathologic; Pathway interactions; Performance; Pharmaceutical Preparations; Pharmacology; Photometry; Physiological; Physiology; Problem Solving; Production; Protein Engineering; Proteins; Public Health; Reaction; Reaction Time; Reactive Oxygen Species; Receptor Activation; Receptor Signaling; Reporter; Resolution; Risk; Rodent; Rodent Model; Second Messenger Systems; Signal Pathway; Signal Transduction; Site; Slice; Specific qualifier value; Specificity; Stress; Structure; System; Time; Toxic effect; United States Dept. of Health and Human Services; Universities; Variant; Ventral Tegmental Area; Washington; Water; addiction; antagonist; biophysical properties; brain tissue; cell type; chromophore; clinically relevant; desensitization; drug development; drug withdrawal; experimental study; in vivo; in vivo fluorescence imaging; in vivo monitoring; innovation; insight; kappa opioid receptors; midbrain central gray substance; motivated behavior; mu opioid receptors; neurotransmission; new technology; novel; opioid epidemic; opioid exposure; opioid overdose; opioid use; prevent; protein expression; real time monitoring; receptor; response; sensor; targeted imaging; tissue preparation; tool

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.

摘要 2017年，美国卫生与公众服务部宣布， 这一年，超过47，000名美国人死于阿片类药物过量。的关键部分 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，这可能有助于发展药物成瘾。近日 已经表明，神经元中μ和κ阿片样物质受体的激活引起反应性阿片样物质的产生， 通过涉及NADPH氧化酶和c-Jun N-末端激酶的途径，因此，我们认为， 这种在受体下游的独特反应可用于检测特异性阿片受体的激活 和调制。目前对阿片受体的研究依赖于细胞培养的体外实验或分析 体外脑组织来监测药物暴露情况。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纤维光度法直接监测μ阿片受体（莫尔） 实时和体内的活性。当代传感器的当前限制是响应时间慢、低响应时间、低响应时间和低响应时间。 特异性、低信号输出、毒性或依赖于离体组织制备。我们的目标是发展一个 一种基因编码的传感器蛋白，可检测内源性ROS水平和响应时间 和信号幅度，其将能够在莫尔激活时体内监测神经元系统。我们 最近通过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敏感性和信号幅度显著地 与其他可用工具相比有所增强。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过氧化氢和过氧化氢之间的界面处进行额外的结构引导蛋白质设计， 所述绿色荧光报告子和所述传感结构域。我们的目标是在体内表达这种新的工具在莫尔 阳性神经元并在药理学上将活性氧信号与莫尔活性联系起来。我们假设活性氧信号 莫尔神经元中的神经元在药物暴露下将增加。其次，我们假设我们将观察到 重复药物暴露下的活性氧瞬变反映了MORs的脱敏。在这个项目结束时， 我们将有一种新型的高度特异性的传感器来监测阿片受体的活性和适应性。我们 这一建议意义重大，因为我们将首次能够在临床上监测这种适应性 阿片类药物暴露的相关信号通路。我们的方法是创新的，因为我们将联合收割机 结构引导的蛋白质工程和阿片类药物触发信号的体内监测， 进入神经元信号通路。此外，这种方法可以广泛应用于未来的研究， 监测药物暴露期间阿片受体的活性水平，并将随后的神经元变化与药物暴露之间的关系联系起来。 信号和可塑性的动机行为，或镇痛耐受性。

项目成果

Live Imaging with Genetically Encoded Physiologic Sensors and Optogenetic Tools.

• DOI: 10.1016/j.jid.2022.12.002
• 发表时间: 2023-03
• 期刊: The Journal of investigative dermatology
• 作者: S. Zaver;C. J. Johnson;Andre Berndt;C. Simpson

Optogenetic Microwell Array Screening System: A High-Throughput Engineering Platform for Genetically Encoded Fluorescent Indicators.

光遗传学微孔阵列筛选系统：基因编码荧光指示剂的高通量工程平台。

• DOI: 10.1021/acssensors.3c01573
• 发表时间: 2023
• 期刊: ACS sensors
• 影响因子: 8.9
• 作者: Rappleye,Michael;Wait,SarahJ;Lee,JustinDaho;Siebart,JamisonC;Torp,Lily;Smith,Netta;Muster,Jeanot;Matreyek,KennethA;Fowler,DouglasM;Berndt,Andre
• 通讯作者: Berndt,Andre

Structure-guided engineering of a fast genetically encoded sensor for real-time H2O2 monitoring.

用于实时 H2O2 监测的快速基因编码传感器的结构引导工程。

• DOI: 10.1101/2024.01.31.578117
• 发表时间: 2024
• 期刊: bioRxiv : the preprint server for biology
• 作者: Lee,JustinDaho;Won,Woojin;Kimball,Kandace;Wang,Yihan;Yeboah,Fred;Evitts,KiraM;Neiswanger,Carlie;Schattauer,Selena;Rappleye,Michael;Bremner,SamanthaB;Chun,Changho;Smith,Netta;Mack,DavidL;Young,JessicaE;Lee,CJustin;Chavki
• 通讯作者: Chavki