Bioluminescent indicators for noninvasive imaging of acetylcholine release

摘要 虽然乙酰胆碱（ACh）分泌神经元只占神经元总数的一小部分，但在神经元中， 在动物大脑中，它们在调节基本大脑功能方面发挥着关键作用。特别是， 胆碱能神经元与阿尔茨海默病（AD）中观察到的神经功能缺损有关， 最常见的衰老神经系统疾病然而，尽管小鼠模型重现了AD的特征， 虽然已经建立，但有效治疗或预防的发展受到我们能力的阻碍， 纵向监测胆碱能功能，并将其与疾病发展过程中的行为变化相关联。 近年来，神经递质的遗传编码荧光指示剂的开发已经取得了进展。 在实时成像活体小鼠神经递质释放方面取得了巨大进展，从而使许多 令人兴奋的发现将特定神经递质的活动与各种行为输出联系起来。但在 脑中的体内荧光成像需要侵入性地插入照明和记录装置， 这很容易造成行为或功能缺陷，特别是当感兴趣区域位于深处时。 因此，需要的是，而不存在的是，一种非侵入性地和纵向地观察释放的方法。 神经递质如乙酰胆碱从动物外部。 我们建议创建和验证乙酰胆碱指示剂，不通过荧光，但通过生物发光， 其中荧光素酶氧化化学底物以在神经递质依赖性细胞中产生光， 方式生物发光成像不需要外部激发光，并且自动生物发光通常是 哺乳动物中缺少的，因此可以很容易地用外部探测器实现深层组织中的灵敏成像。 在我们的初步工作中，我们证明了Anglycerin，一种高催化性和发红光的荧光素酶， 工程，当与我们开发的新型基板相结合时，能够产生55倍的亮度， 与通常使用的萤火虫荧光素酶相比，小鼠大脑中的生物发光。这种最先进的 现在，双磷酸酶-双磷酸酶打开了创造在大脑中发挥作用的敏感生物发光记者的大门。 我们现在建议创建神经递质生物发光指示剂（NeuBI），从ACh开始， 主要目标具体地说，基于蛋白质的ACh指示剂将从荧光素酶Anglycerase开发， （1）细菌ACh结合结构域，或（2）毒蕈碱受体。将对创建的ACh指标进行测试 在培养的神经元和小鼠中成像ACh释放。一旦建立，我们设想这些乙酰胆碱指标将 广泛用于AD小鼠模型中胆碱能活性的非侵入性记录，并可作为模板 用于其他神经递质生物发光指示器的工程设计。

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.