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

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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.