Norepinephrine (NE) ubiquitously mediates cell-to-cell communication, yet NE transmission remains poorly understood. We aim to develop NE sensors to transform NE biological and translational research.
Norepinephrine (NE) is one major neurotransmitterthat mediates cell-to-cell communication in both the central and peripheral nervous systems (CNS and PNS). In CNS, NE is primarily released by the small locuscoeruleus (LC) that innervates a greater varietyof brain areas than any other single nucleus yet described. The broad LC-NE neuronal projection pattern is consistent with itsinvolvement in high cognition, such as attention, behavioral flexibility andsleep/awake states. Indeed,dysregulation of central norepinephrinergic transmission is linked with anumber of neurological, mental and psychiatric diseases, including attentiondeficit disorder, depression,obsessive-compulsivedisorder, sleep disorder,and schizophrenia. In PNS, NE regulatesbiological processes in a variety of other body's organs. Accordingly, aberrant peripheralnorepinephrinergic signaling is associated with many other health problems,including hypertension, obesityand cardiovascular diseases. Despite of itsgeneral functional significances, the precise regulations and roles of centraland peripheralnorepinephrinergic transmissions remain poorly understood, due primarilyto the limited spatial and temporal resolution of the available techniques thatcan non-invasively and efficiently report NE dynamics, which have hindered our understanding the physiologyand pathology of norepinephrinergic transmission. Here, we propose to develop a family ofgenetically-encoded NE sensors, which we expect to transform the related biological and translational research.
We recently engineered a G-protein-coupled receptor activation-basedACh sensor (GACh) by coupling a muscarinic acetylcholinereceptor (MR) with a circularpermutated green fluorescent protein (cpGFP). Using our newly developed high-throughput mutagenesis andscreening method, we refined the sensor with thousands of mutations andtheir combinations, which resulted in a family of ACh sensorswith (EC50 ≈ 1 µM), specificity (comparable to MR), signal-to-noiseratio (SNR ≈ 14), kinetics (ton/off ≈ 200-800 ms) and photostability (≥ 1-4 hrs). GACh sensors were then validated withtransfection, viral and/or transgenic expression in a dozen types of neuronal andnon-neuronal cells prepared from several animal species. In all preparations, GACh sensors selectivelyresponded to exogenous and/or endogenous ACh with robust fluorescence signalsthat were captured by epifluorescent, confocal and/or two-photon microscopy. Moreover, analysis of endogenous ACh releaserevealed, for the first time, firing pattern-dependent release and restrictedvolume transmission, resolving two long-standing questions about centralcholinergic transmission. Thus,we successfully created a user-friendly, broadly applicable toolbox formonitoring cholinergic transmission underlying diverse biological processes. The findings have led to two researcharticles with one published in NatureBiotechnology (2018 Epub online; DOI: 10.1038/nbt.4184) and the other inpreparation for submission to NatureMethods, as well as two NIH awards (BRAINI U01NS103558, for ourcollaborators, and R01NS104670).
Using the same strategy, we initiated development ofgenetically-encoded G-protein-coupled receptor activation-based sensors for NE(GANE) by coupling cpGFP with a norepinephrinereceptor. We are improving the sensorswith large-scale site-directed mutagenesis and screening. Our preliminary data suggest that GANE sensors will have specificity,signal-to-noise ratio, kinetics and photostability suitable for real-time imagingof endogenous NE signals. We will refine the sensors and combine thesensors with optogenetics and various imaging approaches to understand somefundamental questions of norepinephrinergic transmissions in neuronal and non-neuronal cells in vitro and in behaving animals. This proposal will capitalize on the synergistic,but different physical and intellectual assets at the Zhu, Lynch andVenton labs that are essential for the proposed genetics, molecular biology,electrophysiology, imaging, optogenetics, voltammetry and behavior experiments.
We expect the findingsfrom this project to result in multiple publications in high-profile journals,and to win additional NIH awards, including BRAINI and R01 grants. Mostlyimportantly, we expect our GANE sensors to transform the related biological and translational research.
All of the funds will be used for salary supportfor students in the three labs, including Dr. Yajun Zhang, a postdoctoral researchassociate in the Zhu lab, Dr. Tanseli Nesil, a research scientist in the Lynchlab, and Dr. Mimi Shin, a postdoctoral research associate in the Venton lab. The supplies and other costs will becovered by NIH grants from the Zhu,Lynch and Venton labs.