The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 10019971
• 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/10019971
• 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; Behavior; Behavioral; Biological Models; Brain; Brain region; Calcium; Cancer Control; Canis familiaris; Capsaicin; Capsicum; Cell Nucleus; Cell membrane; Cells; Cerebral cortex; Clinical; Clinical Trials; Communication; Communities; Computer software; Data; Data Set; Databases; Defect; Devices; Dissociative Anesthetics; Dorsal; Dynorphins; Environment; Enzymes; Evolution; Exposure to; Family; Fluorescence; Food; Foundations; Ganglia; Gel Chromatography; Gene Expression; Gene Expression Profile; General Anesthesia; General anesthetic drugs; Genes; Genetic Transcription; Glutamates; Goals; High Pressure Liquid Chromatography; High-Throughput Nucleotide Sequencing; Hippocampus (Brain); Human; Human Resources; In Situ Hybridization; Inflammation; Infrastructure; Infusion procedures; Inhalation; Inhalation Anesthetics; Intervention; Investigation; Ion Channel; Isoflurane; Ketamine; Knowledge; Label; Laboratories; Lead; Ligands; Limb structure; Lipids; Measures; Mediating; Memory; Messenger RNA; Methodology; Modeling; Molecular; Molecular Biology; Molecular Neurobiology; Molecular Profiling; Morphine; Motor; Mus; Myelin Sheath; Nerve; Nerve Endings; Neuraxis; Neuroglia; Neurons; Nociception; Nuclear; Opioid; Opioid Peptide; Organism; Pain; Pain Research; Pain management; Pathologic; Pathway interactions; Patients; Peptides; Peripheral; Peripheral Nerves; Persistent pain; Pharmacology; Physiological; Physiology; Population; Posterior Horn Cells; Preparation; Process; Proteins; Protocols documentation; Publishing; Radioimmunoassay; Rattus; Recovery; Regulation; Reporting; Research; Research Methodology; Resiniferatoxin; Resistance; Resolution; Resources; Role; Running; Sampling; Schwann Cells; Scientist; Sensory; Spinal Cord; Spinal Ganglia; Supporting Cell; Surgical incisions; Synapses; Synaptic Transmission; System; TRPV1 gene; Technology; Testing; Time; Tissue Procurements; Tissue Sample; Tissues; Training; Transcript; Transcription Process; Transcriptional Regulation; Up-Regulation; analog; anaplastic lymphoma kinase; cancer pain; chronic pain; clinical pain; cognitive function; cognitive process; cohort; comparative; deep sequencing; dorsal horn; endogenous opioids; experimental study; functional plasticity; human model; human tissue; injured; insight; kappa opioid receptors; mRNA sequencing; neural circuit; non-opioid analgesic; opioid epidemic; pain model; pain patient; paralogous gene; pre-prodynorphin; preproenkephalin; receptor; recruit; relating to nervous system; therapeutic development; transcription factor; 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 resulting in many billions of reads of transcriptome sequence information. We are intensively involved in the analysis of the resulting datasets that encompass physiologically or genetically labeled pain-sensing neurons, neurons in dorsal spinal cord during peripheral inflammation, models of inflamed or surgical incision peripheral tissue, axotomized dorsal root ganglion (DRG) neurons, dorsal and ventral spinal cords, peripheral nerve, and peripheral tissue as well as human tissues comprising the nociceptive circuit. 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 investigation that distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome and 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. This is a high-resolution, transformative technology that provides sequence-based counting of transcripts to categorize, for example, genes that 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 examined the endogenous opioid peptide precursors and peptides preproenkephalin and pre prodynorphin in dorsal spinal cord after an experimental inflammation through the combined use of radioimmunoassay and HPLC and gel filtration chromatography for peptide levels, RNA-Seq for the mRNA levels, and in situ hybridization for combined cellular localization as well as behavioral characterization of the animals alteration in nociceptive behaviors. The focus was on the dynorphin family of endogenous opioids, and this opioid peptide and mRNA was strongly up-regulated in dorsal spinal cord neurons. We also performed a direct comparison of transcriptome level alterations in dorsal horn after inflammation compared to surgical incision. Dynorphin was a strong neuronal signature in both models. Transcriptome analysis also showed upregulation of anaplastic lymphoma kinase (ALK) in the spinal cord sample. In situ hybridization showed that ALK and dynorphin were in the same subpopulation of neurons and that these constituted a population of glutamatergic dorsal horn neurons. Analyses of the kappa opioid receptor system using a selective antagonist to block the action of endogenous dynorphin The results suggest that endogenous dynorphin acts to aid in resolution of the hyperalgesic state engendered by peripheral inflammation. Thus, we identify a critical component, dynorphin, and a critical cell population, dynorphinergic-glutamatergic excitatory dorsal horn neurons that participate in the regulation of spinal cord hyperexcitability. We hypothesize that these neurons are of adaptive significance, such that they reduce tonic hyperexcitability and allow an injured organism to continue to forage for food while protecting the injured limb from further damage. 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 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 general anesthesia can transiently uncouple synaptic activity from neuronal transcriptional control. The situation turned out to be quite different for ketamine where we saw activation of a subset of immediate early transcription factors rather than a suppression of their basal expression as well as activation of the Nrf2 pathway.

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。 We are intensively involved in the analysis of the resulting datasets that encompass physiologically or genetically labeled pain-sensing neurons, neurons in dorsal spinal cord during peripheral inflammation, models of inflamed or surgical incision peripheral tissue, axotomized dorsal root ganglion (DRG) neurons, dorsal and ventral spinal cords, peripheral nerve, and peripheral组织以及包括伤害性电路的人体组织。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）。 We published initial reports on the comprehensive transcriptomic profile of this clinically important population of nociceptive neurons, followed by a second investigation that distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome and 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,一系列患有癌症疼痛的犬队也因RTX疼痛和大鼠受控治疗而受到治疗。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。这是一项高分辨率的变革性技术，可提供基于序列的转录本计数，以分类，例如，与在无关紧要的水平上表达的基因相比，基因表达得很好。此外，我们可以对伤害感受的分子旁系同源物进行定性分配，从而可以出现更有信息的机械框架。 In this cycle we examined the endogenous opioid peptide precursors and peptides preproenkephalin and pre prodynorphin in dorsal spinal cord after an experimental inflammation through the combined use of radioimmunoassay and HPLC and gel filtration chromatography for peptide levels, RNA-Seq for the mRNA levels, and in situ hybridization for combined cellular localization as well as behavioral characterization of动物改变伤害性行为。 重点是内源性阿片类药物的动源家族，这种阿片类肽和mRNA在背脊髓神经元中强烈上调。与手术切口相比，我们还对炎症后背角的转录组水平改变进行了直接比较。在这两种模型中，Dynorphin都是强烈的神经元特征。 转录组分析还表明，脊髓样品中肿瘤淋巴瘤激酶（ALK）的上调。原位杂交表明，ALK和Dynorphin处于神经元的相同亚群中，并且这些构成了谷氨酸甲状腺反性角神经元的种群。 使用选择性拮抗剂来阻断内源性驱体的作用结果表明，内源性测能作用有助于解决外周炎症导致的高过敏状态，对Kappa阿片受体系统进行分析。因此，我们确定了一个关键成分，驱显肽和关键细胞种群，dynorphinophinerphinagric-glutamatorgic兴奋性背角神经元，该神经元参与调节脊髓过度兴奋性。我们假设这些神经元具有适应性的意义，因此它们可以降低强直性过度兴奋性，并允许受伤的生物体继续觅食，同时保护受伤的肢体免受进一步损害。 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。这些是对认知功能大麻醉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，全身麻醉可以从神经元转录控制中瞬时脱离突触活动。对于氯胺酮来说，这种情况是完全不同的，在那里我们看到了立即转录因子的一部分激活，而不是抑制其基础表达以及NRF2途径的激活。