Characterizing the Cocaine-responsive peptidome in mammalian telecephalon

哺乳动物端脑可卡因反应肽组的表征

• 批准号: 9036371
• 负责人: BRIAN A BALDO
• 金额: $ 22.51万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9036371
• 关键词: Acute; Adverse effects; Affinity; Animals; Antibodies; Attention; Behavioral; Biogenic Amines; Biology; Brain; Brain region; Cocaine; Cocaine Dependence; Collection; Coupled; Dependence; Development; Dopamine; Drug Addiction; Drug abuse; Dynorphins; Enkephalins; Extracellular Space; Gases; Gene Expression; Goals; Gold; Grant; Guns; Health; Image; Imaging Techniques; In Situ Hybridization; Injection of therapeutic agent; Intoxication; Label; Leucine; Magnetic nanoparticles; Maps; Mass Spectrum Analysis; Methods; Microdialysis; Morbidity - disease rate; Neuraxis; Neuromodulator; Neurons; Neuropeptides; Nucleus Accumbens; Opioid; Pharmaceutical Preparations; Pharmacotherapy; Phase; Play; Prefrontal Cortex; Procedures; Process; Proteins; Public Health; Recovery; Relapse; Resolution; Rewards; Role; Running; Sampling; Self Administration; Shotguns; Signal Transduction; Site; Slice; Spatial Distribution; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staging; Stimulus; Substance abuse problem; Technology; Testing; Therapeutic; Time; Tissues; Translating; Withdrawal; Work; addiction; adverse outcome; base; behavior test; beta-Endorphin; combinatorial; comparative; design; gene discovery; gene product; improved; innovation; insight; mRNA Expression; mortality; nanoparticle; neurochemistry; next generation; novel; physical symptom; productivity loss; psychological symptom; response; success; tandem mass spectrometry; temporal measurement; theories; transmission process; treatment strategy

 DESCRIPTION (provided by applicant): The public health impact of substance abuse is enormous and widespread; adverse consequences include mortality, morbidity including debilitating physical and psychological symptoms, and loss of productivity. Existing drugs are not completely effective and often characterized by adverse side effects; hence, there is a great need to discover new neurochemical targets for the development of addiction therapeutics. As a neurochemical class, peptides are understudied relative to "classic" neuromodulators such as dopamine. Yet, work on a relatively small number of a priori-identified peptides show that they play profound functional roles across the addiction cycle. The next generation of anti-addiction medications could very conceivably be based on yet undiscovered peptides with specific functional roles in the addiction cycle. In this proposal, we aim to apply a cutting-edge mass spectrometry (MS)-based analytic platform to discover new peptides (peptidomic discovery), and characterize already-known peptides with unprecedented precision, across two drug states: acute cocaine intoxication and peak cocaine withdrawal. The technological platform consists of the following methods: 1. Shotgun peptidomics approach based on gas-phase tandem MS fragmentation methods coupled with isobaric tagging to rapidly quantify a large number of neuropeptides; 2. Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of thin tissue slices using a novel gold-nanoparticle matrix, in correlation with in situ hybridization, to map the spatial distribution of peptide fluxes and enable co-localization wih dopamine signals; 3. Affinity-enhanced microdialysis using innovative magnetic nanoparticles to massively amplify the recovery of peptide efflux from the central nervous system (CNS) extracellular space, thereby improving sensitivity and temporal resolution; 4. Synthesis of any novel peptides discovered, and behavioral testing of these sequences in a well- validated brain stimulation-reward threshold procedure. These methods are highly advanced, and some new innovations have never been tried before in mammalian tissues. Hence, considering the time constraints of the R21 grant mechanism, our first goal is to refine and optimize the technological platform by specifically focusing on simultaneous -opioid, -opioid, and dopamine co-transmission in the nucleus accumbens (Acb) and prefrontal cortex (PFC) during acute cocaine intoxication and peak cocaine withdrawal. This type of combinatorial analysis in both brain sites has never been attempted. Our work has the potential to reveal crucial insights regarding an important opponent-process theory in addiction biology, positing diverse and opposing - opioid actions relative to -opioid/dopamine actions across the addiction cycle. Choosing acute cocaine intoxication and peak withdrawal offers the strongest test of the theory. Insights gained from these analyses could translate into uniquely effective combinatorial treatment strategies. Our second goal is to test any new peptide sequences discovered in the MS-based "shotgun" peptidomic analysis in the subsequent stages of our platform, as time permits. This plan will allow us to refine a powerful, technologically advanced platform for peptidomic discovery, and achieve unprecedented 'vertical integration' from the transcriptional to the behavioral levels. At the same time, our studies will ask a discrete question of great scientific importance regarding the interplay among opioid peptides and dopamine in distinct cocaine-associated states.

 描述（由申请人提供）：药物滥用对公共卫生的影响是巨大和广泛的;不良后果包括死亡率、发病率（包括使人衰弱的身体和心理症状）和生产力丧失。现有的药物并不完全有效，并且通常具有不良副作用;因此，非常需要发现新的神经化学靶点以用于成瘾治疗的发展。作为一种神经化学物质，肽相对于“经典”神经调质如多巴胺而言研究不足。然而，对相对少量的优先识别肽的研究表明，它们在整个成瘾周期中发挥着深远的功能作用。下一代的抗成瘾药物很可能是基于尚未发现的在成瘾循环中具有特定功能作用的肽。在这项提案中，我们的目标是应用一个尖端的基于质谱（MS）的分析平台来发现新的肽（肽组发现），并以前所未有的精度表征已知的肽，跨越两种药物状态：急性可卡因中毒和可卡因戒断高峰。该技术平台主要包括以下几个方面：1.鸟枪肽组学方法基于气相串联质谱裂解方法结合同量异位素标签快速定量大量的神经肽; 2.使用新型金纳米颗粒基质对薄组织切片进行基质辅助激光解吸/电离质谱成像（MALDI-MSI），与原位杂交相关，以绘制肽通量的空间分布并实现与多巴胺信号的共定位; 3.亲和增强微透析使用创新的磁性纳米颗粒，以大规模放大从中枢神经系统（CNS）细胞外空间的肽流出的恢复，从而提高灵敏度和时间分辨率; 4.发现的任何新肽的合成，以及在充分验证的脑刺激-奖励阈值程序中对这些序列的行为测试。这些方法都是非常先进的，而且一些新的创新以前从未在哺乳动物组织中尝试过。因此，考虑到R21赠款机制的时间限制，我们的第一个目标是通过专门关注急性可卡因中毒和可卡因戒断高峰期间中脑核（Acb）和前额叶皮层（PFC）中的β-阿片样物质，β-阿片样物质和多巴胺的同时共传递来完善和优化技术平台。在两个大脑部位进行这种类型的组合分析从未被尝试过。我们的工作有可能揭示有关成瘾生物学中重要的对手过程理论的关键见解，并在整个成瘾周期中提出相对于β-阿片类药物/多巴胺作用的多样化和相反的β-阿片类药物作用。选择急性可卡因中毒和戒断高峰期为理论提供了最有力的检验。从这些分析中获得的见解可以转化为独特有效的组合治疗策略。我们的第二个目标是在时间允许的情况下，在我们平台的后续阶段中测试在基于MS的“鸟枪”肽组分析中发现的任何新的肽序列。该计划将使我们能够完善一个强大的、技术先进的肽组发现平台，并实现从转录到行为水平的前所未有的“垂直整合”。与此同时，我们的研究将提出一个具有重要科学意义的离散问题，即阿片肽和多巴胺在不同可卡因相关状态下的相互作用。