Bioluminescent indicators for noninvasive imaging of acetylcholine release

摘要 尽管分泌乙酰胆碱(ACh)的神经元只占小鼠脑内总神经元的一小部分，但 动物的大脑，它们在调节基本的大脑功能方面发挥着关键作用。特别是，监管的失调 胆碱能神经元与阿尔茨海默病(AD)中观察到的神经缺陷有关， 最常见的衰老神经系统疾病。然而，尽管小鼠模型概括了AD的特征 已经建立，有效治疗或预防的发展受到我们的能力的阻碍 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，神经递质的基因编码荧光指示剂的发展 在活体小鼠体内实时成像神经递质释放方面取得了巨大进展，从而使许多 将特定神经递质的活动与各种行为输出联系起来的令人兴奋的发现。但是，在 大脑中的活体荧光成像需要侵入性地插入照明和记录设备， 这很容易造成行为或功能缺陷，特别是当感兴趣区位于深处时。 因此，所需要的，以及不存在的，是一种非侵入性和纵向观察释放的方式 来自动物外部的神经递质，如ACh。 我们建议创建并验证ACH指示剂，该指示剂不是通过荧光而是通过生物发光来工作的， 其中，荧光素酶氧化化学底物以产生依赖于神经递质的光 举止。生物发光成像不需要外部激发光，通常是自动生物发光 因此，利用外部探测器可以很容易地在深层组织中实现灵敏的成像。 在我们的初步工作中，我们证明了Antares，一种高度催化和发出红色发光的荧光素酶 经过改造的，当与我们开发的新型底物相结合时，能够产生55倍的亮度 小鼠脑内的生物发光，与常用的萤火虫荧光素酶进行了比较。这是最先进的 荧光素酶-荧光素对现在打开了创造灵敏的生物发光记者的大门，这些记者在大脑中发挥作用。 我们现在建议创建神经递质生物发光指示器(NeuBis)，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Antares发展而来，并且 (1)细菌ACh结合区，或(2)M受体。将测试创建的ACH指标 在培养的神经元和小鼠中成像ACh的释放。一旦确定，我们预计这些ACH指标将是 广泛用于非侵入性记录AD小鼠模型的胆碱能活动，并可作为模板 用于设计其他神经递质生物发光指示器。

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.