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. !

摘要 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。 酸(GABA)是大脑中传递快速信息流的物质。这一过程受到神经调节因子的严格调控-- 激光包括单胺类和神经肽类。在神经调节剂中，特别是神经肽，有 很难研究，因为它们通常是化学惰性的(非氧化性)，通常激活7-转氨酶- 膜G蛋白偶联受体(GPCRs)依赖延迟(几秒到几分钟)的第二信使 信号，排除了他们的传统电生理学或氧化探针的研究。此外，Endog- 神经肽通常从大的多蛋白前体中被分类到特定的致密核心池中 小泡(DCV)，这表明需要一种不干扰生物发生和 内源DCV的本机排序。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力学。为了满足这一需求， 我们建议开发一种新的基因编码光学传感器家族，称为神经干细胞嵌合探测器。 多肽释放(CDNRs)，通过利用B类(类分泌素)GPCRs的独特结构特征 唯一识别多肽为其天然配体的公司。基于最近解决的蛋白质结构和 为了应用和验证我们的方法，我们将专门关注葡聚糖- 促肾上腺皮质激素释放激素/因子(CRH/CRF)和促性腺激素释放激素-1(GLP-1)具有重要作用。 影响复杂的神经行为，如进食和应激。CDNRs在神经肽检测中的验证 发布后，我们将使用最先进的病毒介导基因在已定义的GLP-1和CRF电路中表达CDNRs 在特定的转基因小鼠模型中进行转导，并执行高分辨率光学记录以消除 检测内源性GLP-1和CRF在体外脑片和行为动物体内的释放。Suc- 这项工作的成功实施将提供一套新颖且经过良好验证的基因编码的CDNR， 可以立即应用于解剖大脑中GLP-1和CRF的肽能回路。此外，这将是 也为未来发展其他几种神经网络探测器奠定了概念和技术基础。 多肽(PACAP、VIP、CGRP和分泌素)属于B类GPCRs。 好了！