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。

基本信息

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
项目类别：
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

项目摘要

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.

抽象的 2017年，美国卫生与公共服务部宣布正在进行的阿片类药物流行为公众在那一年，超过47,000名美国人死于阿片类药物过量的健康危机。一个关键部分解决方案是了解阿片类药物刺激脑回路的基本反应和适应。为了例如，阿片受体的脱敏是疼痛管理中的关键问题，因为它需要增加剂量的镇痛化合物，这可能有助于发展药物成瘾。最近，它已显示出神经元中Mu和Kappa阿片受体的激活导致反应性产生通过涉及NADPH氧化酶和C-Jun N末端激酶的途径（ROS）。所以，从受体下游的这种独特的反应可用于检测特定的阿片受体激活和调制。当前对阿片类药物受体的研究依赖于细胞培养的体外实验或分析在药物暴露下监测其前体脑组织。我们目前缺乏敏感的荧光传感器，这将使我们能够利用最先进的纤维光度法直接监测Mu-Apoid受体（MOR）实时和体内活动。当代传感器的当前局限性是缓慢的响应时间，低特异性，低信号输出，毒性或对离体组织制备的依赖性。我们的目标是开发遗传编码的传感器蛋白，该蛋白在响应时间内检测内源水平的ROS水平和信号振幅，将在MOR激活后对神经元系统进行体内监测。我们最近通过将绿色荧光蛋白融合到细菌中，开发了一种新型的荧光ROS传感器过氧化氢结合蛋白。信号动力学，ROS灵敏度和信号幅度显着与其他可用工具相比，增强了。我们假设我们可以进一步提高该工具的保真度通过在氢 - 氧化氢结合位点和之间的其他结构引导的蛋白质设计和绿色荧光记者和传感域。我们的目标是在MOR中表达这种新颖的工具阳性神经元，并将ROS信号与MOR活性在药理学上联系起来。我们假设ROS信号在MOR中，神经元将在药物暴露下增加。其次，我们假设我们将观察到减少反复暴露在反映MOR脱敏的药物暴露下的ROS瞬变。在这个项目结束时，我们将拥有一个新颖且高度特异性的传感器，用于监测阿片受体活性和适应性。我们的提案很重要，因为这是我们第一次能够监视该临床的适应在体内接触阿片类药物的相关信号通路。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对阿片类药物触发信号的体内监测，以剖析难以剖析访问神经元信号通路。此外，这种方法可以在以后的研究中广泛应用监测药物暴露期间阿片受体的活性水平，并将神经元随后的变化联系起来对动机行为或镇痛耐受性的信号传导和可塑性。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
作者：
S. Zaver;C. J. Johnson;Andre Berndt;C. Simpson
通讯作者：
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
影响因子：
0
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