Developing genetically-encoded detectors for neuropeptide release based on class B G-protein coupled peptide receptors

开发基于 B 类 G 蛋白偶联肽受体的神经肽释放基因编码检测器

• 批准号: 9805402
• 负责人: ZHIPING P. PANG
• 金额: $ 218.71万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9805402
• 关键词: Address; Animal Behavior; Animal Model; Animals; Behavior; Behavioral Paradigm; Binding; Biogenesis; Biosensor; Brain; Brain region; Butyric Acids; Characteristics; Chemicals; Complex; Corticotropin-Releasing Hormone; Corticotropin-Releasing Hormone Receptors; Coupled; Cultured Cells; Data; Dense Core Vesicle; Detection; Development; Electrophysiology (science); Engineering; Extracellular Domain; Family; Feeding behaviors; Fiber; Foundations; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic; Genetic Models; Glutamates; Goals; Green Fluorescent Proteins; Ligands; Measures; Mediating; Membrane; Methodology; Modeling; Molecular Conformation; Mus; Mutagenesis; N-terminal; Neuromodulator; Neurons; Neuropeptides; Neurotransmitters; Noise; Optics; Outcome; Pattern; Peptide Receptor; Peptides; Photometry; Physiological; Polyproteins; Process; Property; Proteins; Regulation; Resolution; Second Messenger Systems; Secretin; Signal Transduction; Site; Slice; Sorting - Cell Movement; Stress; Structural Protein; Structure; Synaptic Transmission; Time; Transduction Gene; Transmembrane Domain; Validation; Viral; Work; base; cellular imaging; combinatorial; detector; feeding; glucagon-like peptide 1; in vivo; in vivo imaging; innovation; millisecond; monoamine; mouse model; neurobehavior; neurochemistry; novel; optical sensor; pituitary adenylate cyclase activating polypeptide; protein structure; real-time images; receptor; relating to nervous system; release factor; response; screening; sensor; small molecule; tool

Abstract Synaptic transmission is mainly mediated by classical neurotransmitters such as glutamate and g-amino butyric acid (GABA) which transduce fast information flow in the brain. This process is tightly regulated by neuromodu- lators including monoamines and neuropeptides. Among neuromodulators, neuropeptides in particular, have been difficult to study because they are often chemically inert (non-oxidizable) and typically activate 7 transmem- brane G-protein coupled receptors (GPCRs) which rely on delayed (seconds to minutes) second messenger signaling, precluding their study by conventional electrophysiology or with oxidizable probes. Moreover, endog- enous neuropeptides are typically sorted from large polyprotein precursors into specific pools of dense core vesicles (DCVs), suggesting the need for a detection strategy that does not interfere with the biogenesis and native sorting of endogenous DCVs. As a result, there is currently a lack of suitable tools for studying the spatial and temporal dynamics of neuropeptide release and peptidergic neurocircuitry. Toward addressing this need, we propose to develop a new family of genetically-encoded optical sensors, termed Chimeric Detectors for Neu- ropeptide Release (CDNRs), by harnessing the unique structural signatures of Class B (Secretin-like) GPCRs that exclusively recognize peptides as their native ligands. Based on recently solved protein structures and the availability of new robust genetic models to apply and validate our approach, we will specifically focus on gluca- gon-like peptide-1 (GLP-1) and corticotropin-releasing hormone/factor (CRH/CRF) which have important func- tions in complex neurobehaviors such as feeding and stress. To validate CDNRs for detection of neuropeptide release, we will express CDNRs in defined GLP-1 and CRF circuitry using state-of-the-art viral mediated gene transduction in specific genetically modified mouse models and perform high-resolution optical recording to de- tect release of endogenous GLP-1 and CRF, both ex vivo in brain slices and in vivo in behaving animals. Suc- cessful implementation of this work will deliver a set of novel and well validated genetically-encoded CDNRs that can be immediately applied to dissect the peptidergic circuitry of GLP-1 and CRF in the brain. Moreover, this will also lay a conceptual and technical foundation for the future development of detectors for several other neuro- peptides (PACAP, VIP, CGRP and secretin) included among peptide-binding class B GPCRs. !

抽象的 突触传递主要由谷氨酸和 g-氨基丁酸等经典神经递质介导 氨基丁酸 (GABA)，可在大脑中快速传递信息。这个过程受到神经调节剂的严格调控 包括单胺和神经肽。在神经调节剂中，尤其是神经肽， 很难研究，因为它们通常具有化学惰性（不可氧化）并且通常会激活 7 种转膜 膜 G 蛋白偶联受体 (GPCR)，依赖于延迟（数秒至数分钟）的第二信使 信号传导，排除了通过传统电生理学或可氧化探针进行的研究。此外，内源性 内源神经肽通常从大的多蛋白前体中分类到特定的致密核心池中 囊泡（DCV），表明需要一种不干扰生物发生和的检测策略 内源 DCV 的天然分选。因此，目前缺乏合适的工具来研究空间 以及神经肽释放和肽能神经回路的时间动态。为了满足这一需求， 我们建议开发一个新的基因编码光学传感器系列，称为神经嵌合探测器 通过利用 B 类（类分泌素）GPCR 的独特结构特征，释放绳肽 (CDNR) 专门识别肽作为其天然配体。基于最近解决的蛋白质结构和 如果有新的稳健遗传模型来应用和验证我们的方法，我们将特别关注葡萄糖 gon 样肽-1 (GLP-1) 和促肾上腺皮质激素释放激素/因子 (CRH/CRF) 具有重要的功能 复杂的神经行为（例如进食和压力）。验证用于检测神经肽的 CDNR 发布后，我们将使用最先进的病毒介导基因在定义的 GLP-1 和 CRF 电路中表达 CDNR 在特定的转基因小鼠模型中进行转导并进行高分辨率光学记录以消除 内源性 GLP-1 和 CRF 的有效释放，无论是在脑切片中离体还是在行为动物体内。苏克- 这项工作的成功实施将提供一组新颖且经过充分验证的基因编码 CDNR， 可以立即应用于解剖大脑中 GLP-1 和 CRF 的肽能回路。而且，这将 也为未来开发其他几种神经元探测器奠定了概念和技术基础 肽（PACAP、VIP、CGRP 和促胰液素）包含在肽结合 B 类 GPCR 中。 ！