Bioluminescent indicators for noninvasive imaging of acetylcholine release

摘要

ABSTRACT Although acetylcholine (ACh)-secreting neurons only constitute a small fraction of the total neurons in an animal brain, they play a critical role in regulating essential brain functions. In particular, the dysregulation of cholinergic neurons has been connected to the neurological deficits observed in Alzheimer’s disease (AD), the most common neurological disorder of aging. However, while mouse models recapitulating the features of AD have been established, the development of effective therapies or preventions has been hampered by our ability to longitudinally monitor cholinergic function and correlate it to behavioral changes during disease development. In recent years, the development of genetically encoded fluorescent indicators for neurotransmitters has made enormous progress in real-time imaging neurotransmitter release in live mouse, thereby enabling many exciting discoveries relating the activity of specific neurotransmitters to various behavioral outputs. However, in vivo fluorescent imaging in the brain suffers from the need to invasively insert illumination and recording devices, which could easily create behavioral or functional deficits, especially when the region of interest is located deeply. Thus, what is needed, and what does not exist, is a way to non-invasively and longitudinally observe the release of neurotransmitters such as ACh from outside the animal. We propose to create and validate ACh indicators that operate not via fluorescence but via bioluminescence, in which a luciferase enzyme oxidizes a chemical substrate to produce light in a neurotransmitter-dependent manner. No external excitation light is needed for bioluminescent imaging, and auto-bioluminescence is usually missing in mammals, therefore sensitive imaging in deep tissues can be easily achieved with external detectors. In our preliminary work, we demonstrated that Antares, a highly catalytic and red-emitting luciferase we engineered, when coupled with novel substrates that we developed, was able to produce 55-fold brighter bioluminescence in mouse brain, compared to the commonly used firefly luciferase. This state-of-the-art luciferase-luciferin pair now opens the door to create sensitive bioluminescent reporters that function in the brain. We now propose to create neurotransmitter bioluminescent indicators (NeuBIs), starting with ACh as the primary target. Specifically, the protein-based ACh indicator will be developed from the luciferase Antares, and either (1) a bacterial ACh binding domain, or (2) muscarinic receptors. The created ACh indicators will be tested in cultured neurons and mice to image ACh release. Once established, we envision these ACh indicators will be widely used for non-invasive recording of cholinergic activity in mouse models of AD, and can serve as templates for the engineering of other neurotransmitter bioluminescent indicators.