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Deciphering biased agonistic activation of mu-opioid receptor by novel optogenetic hydrogen peroxide sensor

新型光遗传学过氧化氢传感器破译μ阿片受体的偏向激动激活

基本信息

批准号：
10604662
负责人：
Justin Lee
金额：
$ 4.16万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10604662
关键词：
Acute Addictive Behavior Address Analgesics Animals Anxiety Arrestins Behavior Binding Proteins Brain Cells Chronic Clinic Codeine Complex Coupling Dependence Detection Directed Molecular Evolution Disease Dose Drug Addiction Drug Design Drug Exposure Drug Tolerance Drug Withdrawal Symptoms Embryo Emotions Engineering Enhancers Enterobacteria phage P1 Cre recombinase Epidemic Equipment Exhibits Face Fentanyl Funding Future G Protein-Coupled Receptor Signaling GTP-Binding Proteins Goals Green Fluorescent Proteins Health Homologous Gene Human Hydrogen Peroxide In Vitro Injections Kinetics Ligands Link MAPK8 gene Machine Learning Measures Mediating Molecular Molecular Target Monitor Morphine Mus NADPH Oxidase National Institute of Drug Abuse Neurobiology Neurodegenerative Disorders Neuronal Plasticity Neurons Opioid Opioid Analgesics Opioid Receptor Opioid agonist Output Pain Pain management Pathologic Pathway interactions Performance Persons Pharmaceutical Preparations Pharmacology Physiological Adaptation Problem Solving Production Property Protein Activation Pathway Protein Engineering Proteins Public Health Reaction Reaction Time Reactive Oxygen Species Receptor Activation Reporter Research Rodent Model Signal Pathway Signal Transduction Site Slice Source Specificity Stress Structure System Technology Time Tissues Toxic effect United States Dept. of Health and Human Services Universities Validation Ventilatory Depression Ventral Tegmental Area Virus Washington addiction animal tissue base brain tissue cell type clinically relevant desensitization design disease phenotype drug development drug withdrawal fluorescence imaging high throughput screening improved in vivo innovation insight kappa opioid receptors kidney cell motivated behavior mu opioid receptors neurotransmission novel opioid epidemic opioid exposure opioid overdose opioid use optogenetics overdose death peroxiredoxin prevent prototype quantum receptor response sensor side effect spatiotemporal targeted imaging 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 and more than 100,000 people died due to opioid overdose in 2021. 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 causes 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. However, we currently lack sensitive fluorescent sensors, which would allow us to directly monitor pathways downstream from mu-opioid receptor (MOR) activation in real-time. Current limitations of contemporary sensors are slow response time, low specificity, low signal output, low brightness, and toxicity. My 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 monitoring of neuronal systems upon MOR activation in brain tissue. I have developed a novel fluorescent ROS sensor with significantly improved signaling amplitude, sensitivity, and response kinetics compared to previous sensors. I used a newly identified insertion site on OxyR, a bacterial hydrogen peroxide binding protein, that putatively improved allosteric coupling to the fluorescent reporter domain. We will increase the fidelity of this tool with new green fluorescent protein (AausFP1) that exhibits exceptional quantum yield and brightness. I will optimize a new ROS sensor at an unprecedented rate through a multifaceted approach that combines rational, computational, and evolutionary protein engineering. My objective is to express this novel tool in MOR positive neurons and to link ROS signals to MOR activity pharmacologically. I hypothesize that ROS signals in MOR neurons will increase under morphine but not fentanyl through a pathway including c-Jun N-terminal Kinase, Peroxiredoxin 6 and NADPH oxidase. Furthermore, I hypothesize that we will observe a decrease in ROS transients under repeated drug exposure reflecting the desensitization of MORs. At the end, I will have a novel and highly specific sensor for monitoring opioid receptor activity and adaptivity. My 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. My approach is innovative because it combines novel protein engineering and 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 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阿片类药物持续流行