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）：药物滥用对公共健康的影响是巨大且广泛的；不利后果包括死亡、发病（包括使人衰弱的身体和心理症状）以及生产力损失。现有药物并不完全有效，并且常常存在不良副作用；因此，非常需要发现新的神经化学靶点来开发成瘾疗法。作为一类神经化学物质，肽相对于“经典”神经调节剂（如多巴胺）的研究还不够深入。然而，对相对少量的先验鉴定的肽的研究表明，它们在成瘾周期中发挥着深远的功能作用。下一代抗成瘾药物很可能基于尚未发现的在成瘾周期中具有特定功能作用的肽。在本提案中，我们的目标是应用基于质谱（MS）的尖端分析平台来发现新的肽（肽组学发现），并以前所未有的精度表征已知的肽，跨越两种药物状态：急性可卡因中毒和峰值可卡因戒断。该技术平台由以下方法组成： 1. 基于气相串联质谱裂解方法与同量异位标记相结合的鸟枪法肽组学方法，可快速定量大量神经肽； 2. 使用新型金纳米粒子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. 使用创新的磁性纳米粒子进行亲和力增强的微透析，以大量放大从中枢神经系统（CNS）细胞外空间流出的肽的恢复，从而提高灵敏度和时间分辨率； 4. 合成任何发现的新肽，并在经过充分验证的大脑刺激奖励阈值程序中对这些序列进行行为测试。这些方法非常先进，并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资助机制的时间限制，我们的首要目标是完善和优化技术平台，特别关注急性可卡因中毒和可卡因戒断高峰期间伏隔核（Acb）和前额皮质（PFC）中的-阿片类药物、-阿片类药物和多巴胺同时传输。从未尝试过在两个大脑部位进行这种类型的组合分析。我们的工作有可能揭示关于成瘾生物学中重要的对手过程理论的重要见解，提出在整个成瘾周期中相对于 -阿片/多巴胺作用的多样化和相反的 -阿片类药物作用。选择急性可卡因中毒和峰值戒断提供了对该理论最有力的检验。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测试基于 MS 的“鸟枪”肽组分析中发现的任何新肽序列。该计划将使我们能够完善一个强大的、技术先进的肽组学发现平台，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大科学意义的离散问题，即阿片肽和多巴胺在不同的可卡因相关状态下的相互作用。