Bioluminescent indicators for noninvasive imaging of acetylcholine release

用于乙酰胆碱释放无创成像的生物发光指示器

• 批准号: 10196839
• 负责人: Michael Z. Lin
• 金额: $ 43.37万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10196839
• 关键词: Acetylcholine; Address; Aging; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease patient; Alzheimer' s disease therapy; Amyloid; Animals; Antibodies; Behavioral; Binding; Bioluminescence; Brain; Brain Injuries; Calcium; Calcium-Binding Domain; Cells; Chemicals; Choline; Cholinesterase Inhibitors; Cognitive; Coupled; Development; Devices; Disease; Elements; Engineering; Enzymes; Exhibits; Fiber; Firefly Luciferases; Fluorescence; Functional disorder; Future; Gene Mutation; Human; Image; Impaired cognition; Lead; Learning; Light; Lighting; Luciferases; Mammals; Memory; Methods; Microdialysis; Molecular Conformation; Monitor; Mus; Muscarinic Acetylcholine Receptor; Muscarinic Receptor Binding; Neurologic Deficit; Neurons; Neurosciences; Neurotransmitters; Noise; Optics; Output; Oxides; Pathogenesis; Patients; Periodicity; Photons; Play; Positron-Emission Tomography; Preclinical Testing; Prevalence; Prevention; Prosencephalon; Proteins; Reporter; Reporting; Research; Role; Sampling; Scanning; Signal Transduction; Techniques; Technology; Testing; Tissues; Tracer; Work; base; behavior measurement; calcium indicator; cholinergic; cholinergic neuron; cognitive ability; detector; donepezil; effective therapy; fluorescence imaging; functional improvement; improved; in vivo; interest; lens; light emission; luciferin; mouse model; nervous system disorder; neuron loss; neurotransmitter release; non-invasive imaging; novel; novel strategies; optogenetics; prevent; promoter; real-time images; response; single photon emission computed tomography; temporal measurement; transgenic model of alzheimer disease; transmission process

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.

抽象的 尽管乙酰胆碱（ACH）分泌神经元仅占总神经元的一小部分 动物大脑，它们在调节基本大脑功能方面起着关键作用。特别是，失调 胆碱能神经元与阿尔茨海默氏病（AD），胆碱能缺乏症有关 衰老的最常见神经系统障碍。但是，鼠标模型概括了AD的功能 已经建立了，有效的疗法或预防措施的发展受到了我们的能力的阻碍 纵向监测胆碱能功能，并将其与疾病发育过程中的行为变化相关。 近年来，神经递质的一般编码荧光指标的开发具有 实时成像神经递质在活鼠标中释放中取得了巨大进展，从而使许多 令人兴奋的发现将特定神经递质的活性与各种行为输出有关。但是，在 大脑中的体内荧光成像遭受侵入性插入照明和记录设备的需要， 这可以轻松创建行为或功能定义，尤其是当感兴趣的区域被深入定位时。 那是必要的，以及不存在的东西，是一种非侵入性和纵向观察释放的一种方式 神经递质等神经递质，例如来自动物外部的ACH。 我们建议创建和验证不是通过荧光而是通过生物发光的ACH指标， 其中荧光素酶氧化酶是化学底物以在神经递质中产生光的化学底物 方式。生物发光成像不需要外部兴奋光，并且自动生物含量通常为 因此，在哺乳动物中缺少，因此可以通过外部检测器轻松地实现深层组织中的敏感成像。 在我们的初步工作中，我们证明了Antares是一种高度催化和红色发射荧光素酶 加上我们开发的新型底物的设计，能够生产出55倍的亮度 与常用的萤火虫荧光素酶相比，小鼠脑中的生物发光。这个最新的 现在，荧光素酶 - 卢西蛋白对开门，以创建在大脑中起作用的敏感生物发光记者。 现在，我们建议创建神经递质生物发光指标（Neubis），从ACH开始为 主要目标。特别是，基于蛋白质的ACH指标将从荧光素酶Antares和 （1）细菌ACH结合结构域，或（2）毒蕈碱受体。将测试创建的ACH指标 在培养的神经元和小鼠中以图像ACH释放。建立后，我们设想这些ACH指标将是 广泛用于AD小鼠模型中胆碱能活性的非侵入性记录，可以用作模板 用于其他神经递质生物发光指标的工程。

项目成果

Machine learning identifies experimental brain metastasis subtypes based on their influence on neural circuits.

• DOI: 10.1016/j.ccell.2023.07.010
• 发表时间: 2023-09-11
• 期刊: CANCER CELL
• 影响因子: 50.3
• 作者: Sanchez-Aguilera, Alberto;Masmudi-Martin, Mariam;Navas-Olive, Andrea;Baena, Patricia;Hernandez-Oliver, Carolina;Priego, Neibla;Cordon-Barris, Lluis;Alvaro-Espinosa, Laura;Garcia, Santiago;Martinez, Sonia;Lafarga, Miguel;Renacer, Michael Z. Lin;Al-Shahrour, Fatima;de la Prida, Liset Menendez;Valiente, Manuel
• 通讯作者: Valiente, Manuel

An optimized bioluminescent substrate for non-invasive imaging in the brain.

• DOI: 10.1038/s41589-023-01265-x
• 发表时间: 2023-06
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Su, Yichi;Walker, Joel R. R.;Hall, Mary P. P.;Klein, Mark A. A.;Wu, Xiang;Encell, Lance P. P.;Casey, Kerriann M. M.;Liu, Lan Xiang;Hong, Guosong;Lin, Michael Z. Z.;Kirkland, Thomas A. A.
• 通讯作者: Kirkland, Thomas A. A.

Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging.

• DOI: 10.1021/acschembio.1c00549
• 发表时间: 2021-12-17
• 期刊: ACS chemical biology
• 影响因子: 4
• 作者: Liu S;Su Y;Lin MZ;Ronald JA
• 通讯作者: Ronald JA