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：该项目的目标是了解转录组水平上疼痛感应神经元和周围组织的分子生物学，以及急性和慢性疼痛模型以及人类患者或验尸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，建立了硬件和软件的基础设施，形成了协作安排，培训了一组科学家，并支持人员来利用RNA-Seq的方法，即原位杂交和组织采购。我们已经在各种物种和模型中进行了数百种深层测序跑步，并导致了数十亿的转录组序列信息读取。我们深入参与了从生理或遗传标记的疼痛传感神经元的分析，外周炎症期间背侧脊髓中的神经元，发炎的外围组织的模型，轴轴膜化的背部神经节（DRG）神经元（DRG）神经元，脊髓和脊髓菌丝，伴有脑膜，且症状菌丝，以及peripersal corderal corderaver，且症状。我们还正在研究在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采样多个时间点，以遵循每个点的足够读取深度和样品数量的进化和分辨率，分别允许进行详尽的评估和统计比较。由于我们分离了某些神经元和非神经元细胞群，因此我们知道哪些基因在疼痛感应神经元中，哪些主要是非伴侣感应神经元中的基因，例如本体感受性的主要传入剂，以及支持细胞或schwann细胞。通过这种类型的组织和神经元特异性信息，可以大大提高关于疼痛生理学的尖锐假设的能力。现在，我们拥有有关介导DRG和脊髓感觉和运动功能以及髓鞘鞘形成的所有基因的定量信息，这些基因又使我们能够在动物模型和人类中在外围和中枢神经系统中产生，传播，处理和调节疼痛的新水平。 TRPV1转录组：我们组的一个重要重点是表达TRPV1的热，化学，pH-和脂质反应离子通道的DRG神经元的亚群。该离子通道也由辣椒素（热胡椒粉中的活性成分）门控。我们已经证明，有效的辣椒素类模拟神经毒素（RTX）可以控制狗和人类的癌症疼痛，这表明TRPV1+神经元在临床疼痛传播中起着至关重要的作用。由于针对表达TRPV1的DRG神经元的操作的功效，我们对针对TRPV1神经元的小鼠，大鼠，犬和人神经节制剂进行了深度RNA测序（RNA-Seq）。我们发表了有关该临床上重要的伤害性神经元种群全面的转录组概况的初步报告，其次是第二次区分Schwann细胞与神经元对DRG转录组的贡献。在目前的周期中，我们将分析扩展到通过激动剂激活的钙荧光和DRG鉴定的TRPV1+神经元，并在尸检时从我们的一名人类癌症疼痛患者中获得的DRG，这些患者接受了RTX治疗，RTX治疗，这是一种患有癌症疼痛的犬类群，该患者还接受了RTX疼痛的治疗，并在RTX中治疗了RTX和受控治疗。结合起来，这些数据表明，最敏感的神经元成分是包含TRPV1的中心突出轴突，而DRG中的细胞体对RTX具有相对抗性。从转录组分析中获得了这种重要的机械洞察力，并用于在我们的人类临床试验中微调管理方案。 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。介导已知镇痛药的作用的确切基因（例如吗啡，可乐定，利多卡因，布洛芬，加巴喷丁，加巴喷丁）和新兴的伤害感受器 - 神经元特异性钠通道之间的定量关系。这是一项高分辨率变革性技术，可提供基于测序的转录本计数，以创建基因表达良好的标准，而在无关紧要的水平上表达的标准。此外，我们可以进行定性分配，以了解伤害性人群中哪些分子旁系同源物，从而可以出现更具信息的机械框架。 在这个周期中，我们确定了一种蛋白质酪氨酸激酶和孤儿GPCR，在伤害性人群中表达和上调。我们发表了一份报告，该报告始于脂质生成基因的转录组分析，以定义代谢途径，以生产潜在的神经活性脂质。这种新颖的方法预测了我们使用质谱法验证的两种新脂质。相关的动物行为和人类头痛和牛皮癣研究表明在疼痛敏化和瘙痒中作用。 遗传变异引起的人类疼痛敏感性的转录组学：RNA-SEQ数据为正在进行的研究提供了一种方法，并为我们的所有假设驱动的研究提供了信息。我们正在表征人类遗传拷贝数变化（CNV），导致疼痛不敏感。可以使用CNV的大鼠或小鼠模型来部分探测这些机制。例如，我们能够使用半骨骼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，转录组分析允许更精确的假设产生和检验。 麻醉转录组：我们正在完成对吸入大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的皮质和海马转录组以及相关蛋白质的影响的转录组。这些是对全身麻醉对认知功能的影响进行更大研究的初步步骤。在人类中，全身麻醉可能对认知功能有害。我们假设可以通过了解由麻醉和恢复能力引起的分子级变化来获得对缺陷状态的机理见解。我们的结果表明，全身麻醉强烈抑制突触输入和核转录控制之间的通信，并且在皮层中的改变比海马更明显。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中的相应降低。数据表明，麻醉可以从神经元转录控制中瞬时脱离突触活动。 摘要：在过去的几年中，获取的数据集提供了前所未有且极为细粒度的细节，涉及疼痛感应电路和对麻醉敏感的大脑区域中的基因表达。基本目标是了解我们如何感知和控制痛苦。我们正在准确确定哪种分子是什么类型的疼痛感应神经元以及它们如何共同完成工作的分子。我们已经将这种方法扩展到麻醉剂的作用，并观察到与海马相比，脑皮质对气态麻醉药的敏感性显着。这表明执行功能和工作记忆将是受全身麻醉影响的最易感变量。总而言之，我们的数据为疼痛研究和麻醉群落提供了一种变革性的新资源，并将允许对实验和临床结果进行更精确的评估和验证。