The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 9792184
• 负责人: Andrew Mannes
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9792184
• 关键词: Absence of pain sensation; Acute Pain; Address; Affect; Afferent Neurons; Agonist; Analgesics; Anesthesia procedures; Anesthetics; Animal Model; Animals; Autopsy; Axon; Base Sequence; Behavioral; Biological Models; Brain; Brain region; Calcium; Cancer Control; Canis familiaris; Capsaicin; Capsicum; Cell Nucleus; Cells; Cerebral cortex; Clinical; Clinical Trials; Clonidine; Communication; Communities; Computer software; Copy Number Polymorphism; Data; Data Set; Databases; Defect; Devices; Dissociative Anesthetics; Dorsal; Environment; Enzymes; Evolution; Exposure to; Fluorescence; Foundations; G-Protein-Coupled Receptors; Ganglia; Gene Expression; Gene Expression Profile; General Anesthesia; General anesthetic drugs; Generations; Genes; Genetic Transcription; Genetic Variation; Goals; Grain; Headache; High-Throughput Nucleotide Sequencing; Hippocampus (Brain); Human; Human Genetics; Human Resources; Ibuprofen; In Situ Hybridization; Inflammation; Infusion procedures; Inhalation; Inhalation Anesthetics; Intervention; Investigation; Ion Channel; Ketamine; Knowledge; Label; Laboratories; Lead; Lidocaine; Ligands; Lipids; Mass Spectrum Analysis; Measures; Mediating; Memory; Messenger RNA; Metabolic Pathway; Methodology; Modeling; Molecular; Molecular Biology; Molecular Neurobiology; Molecular Profiling; Morphine; Motor; Mus; Myelin Sheath; Nerve; Nerve Endings; Neuraxis; Neuroglia; Neurons; Nociception; Nociceptors; Nuclear; Occupations; Opioid; Orphan; Pain; Pain Research; Pain management; Paper; Pathologic; Pathway interactions; Patients; Peripheral; Peripheral Nerves; Pharmacology; Physiological; Physiology; Population; Preparation; Process; Production; Protein Tyrosine Kinase; Proteins; Protocols documentation; Pruritus; Psoriasis; Publishing; Rattus; Recovery; Regulation; Reporting; Research; Research Infrastructure; Research Methodology; Resiniferatoxin; Resistance; Resolution; Resources; Rodent Model; Role; Running; Sampling; Schwann Cells; Scientist; Sensory; Sensory Ganglia; Short-Term Memory; Sodium Channel; Spinal Cord; Spinal Ganglia; Supporting Cell; Synapses; Synaptic Transmission; TRPV1 gene; Technology; Testing; Time; Tissue Procurements; Tissue Sample; Tissues; Training; Transcript; Transcription Process; Transcriptional Regulation; Work; analog; cancer pain; chronic pain; clinical pain; cognitive function; cognitive process; cohort; comparative; deep sequencing; executive function; experimental study; functional plasticity; gabapentin; human model; insight; mouse model; neural circuit; novel strategies; opioid epidemic; pain model; pain patient; pain sensitivity; paralogous gene; peripheral pain; receptor; recruit; relating to nervous system; therapeutic development; time use; transcriptome; transcriptome sequencing; transcriptomics; transmission process

Overview: The objectives of this project are to understand the molecular biology of pain-sensing neurons and peripheral tissues at the transcriptome level and modulation of transcriptomic parameters in acute and chronic pain models and in human patients or post-mortem samples. The laboratory has established research methodology and protocols, built an infrastructure of hardware and software, formed collaborative arrangements, trained a team of scientists and support personnel to utilize the methodology of RNA-Seq, in situ hybridization and tissue procurement. We have performed hundreds of deep sequencing runs in various species and models and resulting in many billions of reads of transcriptome sequence information. We are intensively involved in the analysis of the resulting datasets from physiologically or genetically labeled pain-sensing neurons, neurons in dorsal spinal cord during peripheral inflammation, models of inflamed peripheral tissue, axotomized dorsal root ganglion (DRG) neurons, dorsal and ventral spinal cords, peripheral nerve, and inflamed tissue. We are also investigating transcriptional processes affected by general anesthesia in higher order brain regions. Use of the newer high-throughput sequencing devices allows sampling of multiple time points to follow the evolution and resolution of the intervention with enough read depth and number of samples at each point to permit thorough assessment and statistical comparison, respectively. Because we isolated certain neuronal and non-neuronal cell populations, we know which genes are in pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents, and supporting cells or Schwann cells. The ability to form incisive hypotheses regarding pain physiology is greatly advanced by this type of tissue and neuron-specific information. We now have quantitative information on all the genes that mediate DRG and spinal cord sensory and motor functions and formation of the myelin sheath which, in turn, permits us to build new levels of understanding of how pain is generated, transmitted, processed and modulated in the peripheral and central nervous systems in animal models and humans. TRPV1 Transcriptome: One important focus for our group is the subpopulation of DRG neurons that express the thermo-, chemo-, pH-, and lipid-responsive ion channel called TRPV1. This ion channel is also gated by capsaicin, the active ingredient in hot pepper. We have demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in dogs and humans indicating a crucial role for TRPV1+ neurons in transmission of clinical pain. Because of the efficacy of manipulations aimed at the TRPV1-expressing DRG neurons, we performed deep RNA sequencing (RNA-Seq) on mouse, rat, canine, and human ganglionic preparations targeting TRPV1 neurons. We published initial reports on the comprehensive transcriptomic profile of this clinically important population of nociceptive neurons, followed by a second that distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome. In the present cycle we have extended the analysis to TRPV1+ neurons functionally identified by agonist-activated calcium fluorescence and DRGs obtained at autopsy from one of our human cancer pain patients who had been treated with RTX, a cohort of canines with cancer pain that were also treated for pain with RTX and controlled treatments in the rat. In combination, these data demonstrate that the most sensitive neuronal component is the centrally projecting axons that contain TRPV1 whereas the cell bodies in DRG are comparatively resistant to RTX. This important mechanistic insight was gained from transcriptomic analyses and is being used to fine-tune the administration protocol in our human clinical trial. Analgesia transcriptome: One of the most interesting aspects of the transcriptome analyses is quantitative insight provided by next-gen RNA-Seq. The quantitative relationships between the exact genes that mediate actions of known analgesic drugs such as morphine, clonidine, lidocaine, ibuprofen, gabapentin and emerging nociceptor-neuron-specific sodium channels. This is a high resolution transformative technology that provides sequenced based counting of transcripts to create criteria for which genes are well-expressed versus those expressed at an inconsequential level. Additionally we can make qualitative assignments as to which molecular paralogs are in the nociceptive populations allowing a more informative mechanistic framework to emerge. In this cycle we identified a protein tyrosine kinase and an orphan GPCR that are well expressed and upregulated in the nociceptive population. We published a report that began with transcriptomic profiling of lipid-generating genes to define metabolic pathways for production of potentially neuroactive lipids. This novel approach predicted two new lipids which we verified using mass spectrometry. Correlative animal behavioral and human headache and psoriasis studies suggest roles in pain sensitization and itch. Transcriptomics of human pain sensitivity resulting from genetic variations: The RNA-Seq data provides a means for amplification of ongoing studies and informs all of our hypothesis-driven studies. We are in the process of characterizing human genetic copy number variations (CNVs) that produce pain insensitivity. The mechanisms can be probed, in part, using rat or mouse models of the CNVs. For example, we were able to evaluate the integrity of neurons in sensory ganglion and spinal cord using a rodent model of the hemideletion. In these studies, transcriptomic profiling allows for more precise hypothesis generation and testing. Anesthesia Transcriptome: We are in the process of completing a transcriptomic assessment of the effects of inhalation general anesthesia and ketamine infusion on cortical and hippocampal transcriptomes and associated proteins identified from the gene analysis. These are initial steps to a larger investigation of the impact of general anesthesia on cognitive function. In humans, general anesthesia can be deleterious to cognitive function. We hypothesize that mechanistic insight into the defect state can be obtained by understanding the molecular-level changes induced by anesthesia and the capacity for recovery. Our results indicate that communication between synaptic input and nuclear transcriptional control is strongly inhibited by general anesthesia and that the alterations are more pronounced in cortex than hippocampus. We detect widespread modulation of genes that mediate functional plasticity and memory formation. Corresponding decreases in several of the proteins are also observed. The data suggest that anesthesia can transiently uncouple synaptic activity from neuronal transcriptional control. Summary: The datasets acquired over the past several years provide unprecedented and extremely fine-grained detail on gene expression in pain-sensing circuits and anesthesia-sensitive brain regions. The basic goal is to understand how we sense and control pain. We are determining exactly what molecules the different types of pain-sensing neurons make and how they work together to do their job. We have extended this approach to actions of anesthetic agents and observe a remarkable sensitivity of cerebral cortex to gaseous anesthetics compared to hippocampus. This suggests that executive function and working memory will be the most susceptible variables affected by general anesthesia. Taken together our data provide a transformative new resource for the pain research and anesthesia communities, and will allow more precise assessment and verification of experimental and clinical results.

概述：该项目的目标是在转录组水平上了解痛觉神经元和外周组织的分子生物学，以及急性和慢性疼痛模型以及人类患者或死后样本中转录组参数的调节。该实验室建立了研究方法和协议，建立了硬件和软件基础设施，形成了合作安排，培训了一支科学家和支持人员团队，以利用RNA-Seq、原位杂交和组织采购的方法。我们已经在不同物种和模型中进行了数百次深度测序，产生了数十亿次转录组序列信息读取。我们深入参与对生理或基因标记的痛感神经元、外周炎症期间背脊髓神经元、发炎外周组织模型、轴突背根神经节 (DRG) 神经元、背侧和腹侧脊髓的数据集的分析、周围神经和发炎组织。我们还在研究高阶大脑区域受全身麻醉影响的转录过程。使用较新的高通量测序设备可以对多个时间点进行采样，以跟踪干预的演变和解决方案，每个点都有足够的读取深度和样本数量，以便分别进行彻底的评估和统计比较。因为我们分离了某些神经元和非神经元细胞群，所以我们知道哪些基因位于痛觉神经元中，哪些基因主要位于非痛觉神经元中，例如本体感觉初级传入神经元和支持细胞或施万细胞。这种类型的组织和神经元特异性信息极大地提高了形成有关疼痛生理学的深入假设的能力。我们现在掌握了介导 DRG 和脊髓感觉和运动功能以及髓鞘形成的所有基因的定量信息，这反过来又使我们能够对疼痛如何产生、传播、处理和调节建立新的理解水平。动物模型和人类的周围和中枢神经系统。 TRPV1 转录组：我们小组的一个重要关注点是 DRG 神经元亚群，它们表达称为 TRPV1 的热、化学、pH 和脂质响应离子通道。该离子通道也受到辣椒素（辣椒中的活性成分）的控制。我们已经证明，有效的辣椒素类似物树脂毒素 (RTX) 可以控制狗和人类的癌症疼痛，表明 TRPV1+ 神经元在临床疼痛传递中发挥着至关重要的作用。由于针对表达 TRPV1 的 DRG 神经元的操作的有效性，我们对针对 TRPV1 神经元的小鼠、大鼠、犬和人类神经节制剂进行了深度 RNA 测序 (RNA-Seq)。我们发表了关于这一临床上重要的伤害性神经元群体的综合转录组学概况的初步报告，随后发表了第二份报告，区分了施万细胞与神经元对 DRG 转录组的贡献。在本周期中，我们将分析扩展到 TRPV1+ 神经元，这些神经元通过激动剂激活的钙荧光和 DRG 进行功能鉴定，这些神经元是在我们的一位人类癌症疼痛患者的尸检中获得的，该患者曾接受过 RTX 治疗，这是一组患有癌症疼痛的犬科动物，这些患者也接受了 RTX 治疗。使用 RTX 治疗大鼠疼痛并进行对照治疗。综合起来，这些数据表明最敏感的神经元成分是包含 TRPV1 的中央突出轴突，而 DRG 中的细胞体对 RTX 相对具有抵抗力。这一重要的机制见解是从转录组分析中获得的，并用于微调我们的人体临床试验中的给药方案。 镇痛转录组：转录组分析最有趣的方面之一是下一代 RNA-Seq 提供的定量见解。介导已知镇痛药物（如吗啡、可乐定、利多卡因、布洛芬、加巴喷丁）作用的确切基因与新兴伤害感受器神经元特异性钠通道之间的定量关系。这是一种高分辨率的转化技术，可提供基于测序的转录本计数，以创建基因表达良好与表达水平无关紧要的标准。此外，我们可以对伤害感受群体中哪些分子旁系同源物进行定性分配，从而形成信息更丰富的机制框架。 在此周期中，我们鉴定了一种蛋白酪氨酸激酶和一种孤儿 GPCR，它们在伤害感受群体中表达良好且上调。我们发表了一份报告，首先对脂质生成基因进行转录组分析，以确定产生潜在神经活性脂质的代谢途径。这种新颖的方法预测了两种新的脂质，我们使用质谱法对其进行了验证。相关的动物行为和人类头痛和牛皮癣研究表明其在疼痛敏化和瘙痒中的作用。 遗传变异引起的人类疼痛敏感性的转录组学：RNA-Seq 数据提供了一种放大正在进行的研究的方法，并为我们所有假设驱动的研究提供信息。我们正在研究导致疼痛不敏感的人类基因拷贝数变异（CNV）的特征。可以使用大鼠或小鼠的 CNV 模型来部分探究这些机制。例如，我们能够使用半缺失的啮齿动物模型评估感觉神经节和脊髓中神经元的完整性。在这些研究中，转录组分析可以实现更精确的假设生成和测试。 麻醉转录组：我们正在完成吸入全身麻醉和氯胺酮输注对皮质和海马转录组以及从基因分析中鉴定出的相关蛋白的影响的转录组学评估。这些是对全身麻醉对认知功能影响进行更大规模调查的初步步骤。对于人类来说，全身麻醉可能会损害认知功能。我们假设可以通过了解麻醉引起的分子水平变化和恢复能力来获得对缺陷状态的机械洞察。我们的结果表明，突触输入和核转录控制之间的通讯受到全身麻醉的强烈抑制，并且这种变化在皮层中比海马中更明显。我们检测到介导功能可塑性和记忆形成的基因的广泛调节。还观察到几种蛋白质的相应减少。数据表明麻醉可以暂时将突触活动与神经元转录控制分开。 摘要：过去几年获得的数据集提供了关于疼痛感知回路和麻醉敏感大脑区域的基因表达的前所未有的、极其精细的细节。基本目标是了解我们如何感知和控制疼痛。我们正在确定不同类型的疼痛感知神经元产生什么分子以及它们如何协同工作来完成其工作。我们将这种方法扩展到麻醉剂的作用，并观察到与海马相比，大脑皮层对气体麻醉剂的显着敏感性。这表明执行功能和工作记忆将是最容易受到全身麻醉影响的变量。总而言之，我们的数据为疼痛研究和麻醉界提供了变革性的新资源，并将允许对实验和临床结果进行更精确的评估和验证。