The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 9353640
• 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/9353640
• 关键词: Absence of pain sensation; Acute Pain; Address; Advanced Malignant Neoplasm; Affect; Agonist; Analgesics; Anesthesia procedures; Animal Model; Animals; Autopsy; Brain region; Breathing; Calcium; Canis familiaris; Capsaicin; Capsicum; Cells; Cereals; Clinical; Clinical Trials; Clonidine; Communication; Communities; Computer software; Control Animal; Data; Data Set; Databases; Defect; Diphtheria Toxin; Dorsal; Environment; Enzymes; Evolution; Fluorescence; G-Protein-Coupled Receptors; Ganglia; Gene Expression; Gene Expression Profile; General Anesthesia; Generations; Genes; Genetic; Genetic screening method; Goals; Hippocampus (Brain); Histocompatibility Testing; Human; Human Resources; Ibuprofen; Inflammation; Inhalation Anesthetics; Intervention; Intractable Pain; Investigation; Ion Channel; Knowledge; Label; Laboratories; Lidocaine; Ligands; Lipids; Malignant Bone Neoplasm; Measurement; Mediating; Memory; Messenger RNA; Methodology; Methods; Microinjections; Modeling; Molecular; Molecular Neurobiology; Morphine; Motor; Mus; Myelin Sheath; National Institute on Alcohol Abuse and Alcoholism; Nerve Endings; Neuronal Plasticity; Neurons; Nociception; Nociceptors; Nuclear; Occupations; Orphan; Pain; Pain Research; Pain management; Paper; Pathway interactions; Patients; Peripheral; Peripheral Nerves; Peripheral Nervous System; Persistent pain; Pharmaceutical Preparations; Physiological; Physiology; Population; Preparation; Process; Property; Protocols documentation; RNA; Rattus; Reading; Recovery; Recruitment Activity; Regulation; Reproducibility; Research; Research Infrastructure; Research Methodology; Resiniferatoxin; Resolution; Resources; Rheumatoid Arthritis; Role; Running; Sampling; Schwann Cells; Scientist; Sensory; Sensory Ganglia; Signal Transduction; Skin; Sodium Channel; Sorting - Cell Movement; Spinal Cord; Spinal Ganglia; Stimulus; Supporting Cell; Synapses; TRPV1 gene; Techniques; Therapeutic; Time; Tissue Sample; Tissues; Training; Transcript; Transcriptional Regulation; Transducers; Transgenic Organisms; Vulnerable Populations; Work; aged; analog; cancer pain; cell killing; chronic pain; clinical practice; cognitive function; deep sequencing; differential expression; diphtheria toxin receptor; drug development; gabapentin; human data; human tissue; in vivo; insight; interest; killings; lipid transport; meetings; next generation sequencing; osteosarcoma; paralogous gene; peripheral pain; receptor; receptor binding; relating to nervous system; research study; statistics; time use; transcriptome; transcriptome sequencing; transcriptomics; translational study; transmission process

Overview: 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. We have performed over several hundreds of deep sequencing runs on Illumina HiSeq machines and obtained over 20 billion reads of transcriptome sequence information and are intensively involved in the analysis of the resulting datasets. We have sequenced the transcriptomes of physiologically or genetically labeled pain-sensing neurons after isolation by FACS, neurons in dorsal spinal cord during peripheral inflammation, models of rheumatoid arthritis, inflamed peripheral tissue, axotomized DRG, dorsal and ventral spinal cords and peripheral nerve. We have also begun investigations into transcriptional processes affected by general anesthesia in higher order brain regions. In many cases multiple time points are sampled to follow the evolution and resolution of the intervention with enough samples at each point to permit statistical comparison. Because we sorted for certain neuronal populations we know which genes are in pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents, 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 in the peripheral nervous system. 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. Previous experiments demonstrated that the potent capsaicin analog resiniferatoxin (RTX) can control cancer pain in dogs and humans indicating a crucial role for these neurons in transmission of clinical pain. Because of the efficacy of manipulations aimed at the TRPV1-expressing DRG neurons we want to know everything possible about them. We have performed deep sequencing of the mRNA content using next-gen RNA-Seq on various TRPV1 neuronal preparations. A genetic method expresses a fluorescent marker allowing the TRPV1 DRG neurons to be isolated by FACS. To obtain the inverse population, the TRPV1 neurons were killed by making them express diphtheria toxin receptor. Another strategy was to stimulate TRPV1 neurons with RTX and sort the neurons that display increases in calcium fluorescence. We are also killing the cells by microinjection of RTX in vivo. Our first paper outlines the transcriptome results from the genetically labeled TRPV1 neurons and ganglia in which the TRPV1 neurons had been deleted by expression of diphtheria toxin or microinjection of RTX. This has provided comprehensive, new transcriptomic information on genes expressed by a clinically important population of nociceptive neurons. We are now making comparisons of DRGs obtained post-mortem from a cancer pain patient that had been treated with RTX to identify the pain relevant molecular transducers in humans. Analgesia transcriptome: One of the most interesting aspects of the transcriptome analyses is quantitative insight provided by next-gen RNA-Seq. We now know the quantitative relationships between the exact genes that mediate the actions of known analgesic drugs such as morphine, clonidine, lidocaine, ibuprofen, and gabapentin and emerging targets such as nociceptor-neuron-specific sodium channels. Frequently it is not clear which molecular paralogs of ion channels or drug binding receptors are expressed by different tissues in the pain pathway. Our data show that when expression values for all the relevant genes are obtained quantitatively, at the same time, and with excellent reproducibility between animals and treatments a new, more informative picture emerges. The transcriptome experiments also point to new targets for potential analgesic drug development. We identified an orphan GPCR that is well expressed in the nociceptive population, and are currently exploring its analgesic properties. Transcriptomics of Peripheral Sensitization: The RNA-Seq data provides a means for amplification of ongoing studies. The RNA-Seq results inform all of our hypothesis-driven studies and those of other groups. An example is our collaborative work with NIAAA. We observe that certain lipids are TRPV1 agonists. Using the transcriptome databases, we have extracted the quantitative expression data for all the genes involved in lipid transport, generation, degradation, and the cognate receptors for the relevant lipids from sequencing of skin, DRG and dorsal spinal cord. Differential expression levels therein provided insight into new enzymes that generate particular lipids important for nociceptive sensitization. It is noteworthy that this molecular predictive approach has identified totally new groups of endogenous neuro-active lipids. Canine Ganglionic Transcriptome: This year we completed the extraction and sequencing of canine ganglion and spinal cord tissue from controls and animals with osteosarcoma that were euthanized because of inadequate pain control. Tissues were obtained at autopsy. This study was undertaken to test for genes activated by nociceptive input from naturally occurring bone cancer. Some animals also had their pain treated with RTX. This will form a unique dataset that will provide new insight into the transcriptomics of cancer pain in a species that is very similar to humans and the therapeutic actions of RTX. Anesthesia Transcriptome: Another project we are in the process of completing is an assessment of the effects of inhalation general anesthesia on cortical and hippocampal transcriptome. This is the initial step to a larger study on the effects of general anesthesia on cognitive function in aged animals. In humans, general anesthesia can have a deleterious impact on cognitive function and we hypothesize that we can gain mechanistic insight into the defect state by understanding the molecular level changes in gene expression induced by anesthesia and the capacity for recovery. Our initial results indicate that communication between neuronal synaptic input and nuclear transcriptional control is altered by general anesthesia and that the alterations are more pronounced in cortex than hippocampus. It is quite remarkable that we detect widespread modulation of genes that mediate plasticity of neuronal function and memory formation. Summary: The datasets acquired over the past several years provide unprecedented and extremely fine-grained detail on gene expression in pain-sensing circuits. This may seem complicated but the basic goal is to understand how we sense pain and how we may control it when required. There are a wide variety of painful stimuli that can be encountered in our environment and different neurons exist to sense these different types of pain signals. We are determining exactly what molecules the different types of pain-sensing neurons make and how they work together to do their job. We will use this information to understand pain signaling and how to control it. Taken together these data provide a transformative new resource for the pain research community and will allow a much more precise assessment of experimental manipulations and verification of experimental results.

概述：实验室已经建立了研究方法和协议，建立了硬件和软件的基础架构，形成了协作安排，培训了一个科学家团队，并支持人员利用RNA-Seq的方法论。我们已经在Illumina Hiseq机器上进行了数百多个深度测序运行，并获得了超过200亿个转录组序列信息的读取，并强烈参与了所得数据集的分析。我们已经对FACS分离后的生理或遗传标记的疼痛神经元的转录组进行了测序，外周炎症期间背侧脊髓中的神经元，类风湿关节炎模型，发炎的外周血组织，轴轴向DRG，背部脊髓索和脊柱脊髓索和外侧脊髓和外围神经。我们还开始研究在高阶大脑区域受全身麻醉影响的转录过程。在许多情况下，对多个时间点进行了采样，以遵循每个点在每个点上用足够的样品进行干预的演变和解决方案，以允许统计比较。因为我们为某些神经元群体进行了分类，所以我们知道哪些基因在疼痛感应神经元中，哪些主要是非pain感应神经元中的基因，例如本体感受的主要传入剂，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的所有基因的定量信息，以及在周围神经系统中髓鞘鞘的形成。 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。先前的实验表明，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，表明这些神经元在临床疼痛传播中至关重要。 由于针对表达TRPV1的DRG神经元的操作有效性，因此我们想了解它们的所有可能。我们已经在各种TRPV1神经元制剂上使用下一代RNA-Seq对mRNA含量进行了深入测序。遗传方法表达荧光标记物，允许FACS分离TRPV1 DRG神经元。 为了获得逆种群，TRPV1神经元通过使它们表达白喉毒素受体而杀死。 另一种策略是用RTX刺激TRPV1神经元，并对钙荧光增加的神经元进行排序。我们还通过在体内的RTX的显微注射来杀死细胞。我们的第一篇论文概述了转录组由遗传标记的TRPV1神经元和神经节引起的，其中TRPV1神经元通过表达毒素的表达或RTX的显微注射而被删除。这为临床上重要的伤害性神经元群体表达的基因提供了全面的新转录组。 现在，我们正在比较从癌症疼痛患者中获得的DRG，该患者已接受RTX治疗，以识别人类中相关的疼痛相关分子传感器。 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。现在，我们知道介导已知镇痛药的作用（例如吗啡，可乐定，利多卡因，布洛芬和加巴喷丁）的确切基因之间的定量关系，以及诸如Nocteptor-Neuron特异性钠通道等新兴靶标。 通常，尚不清楚哪些分子旁系同源于离子通道或药物结合受体在疼痛途径中的不同组织表达。 我们的数据表明，当所有相关基因的表达值同时获得，并且在动物和治疗之间具有出色的可重复性时，就会出现新的，更有用的图片。转录组实验还指出了潜在的镇痛药物发育的新靶标。我们确定了在伤害性人群中表达很好的孤儿GPCR，目前正在探索其镇痛性能。 周围敏化的转录组学：RNA-seq数据提供了一种正在进行的研究的方法。 RNA-seq结果为我们的所有假设驱动的研究和其他群体的研究提供了信息。一个例子是我们与NIAAA的合作工作。 我们观察到某些脂质是TRPV1激动剂。使用转录组数据库，我们从脂质转运，产生，降解和同源受体中提取了来自皮肤，DRG和背脊髓的测序中相关脂质的所有涉及的基因的定量表达数据。其中的差异表达水平提供了对新酶产生特定脂质对伤害感受敏化重要的新酶的见解。值得注意的是，这种分子预测方法已经确定了全新的内源性神经活性脂质组。 犬神经节转录组：今年，我们从对照组和骨肉瘤的动物中完成了犬神经节和脊髓组织的提取和测序，由于疼痛控制不足，它们被安乐死。尸检时获得组织。这项研究是为了测试来自天然存在的骨癌的伤害性输入而激活的基因。 一些动物还用RTX治疗了疼痛。 这将形成一个独特的数据集，该数据集将为与人类非常相似的物种和RTX的治疗作用提供新的见解。 麻醉转录组：我们正在完成的另一个项目是评估吸入大麻醉对皮质和海马转录组的影响。 这是对大麻醉对老年动物认知功能影响的更大研究的第一步。 在人类中，全身麻醉可以对认知功能产生有害影响，我们假设我们可以通过了解由麻醉和恢复能力引起的基因表达的分子水平变化来获得对缺陷态的机械洞察力。 我们的最初结果表明，神经元突触输入与核转录控制之间的通信会因全身麻醉而改变，并且在皮质中的改变比海马更明显。 非常值得注意的是，我们检测到介导神经元功能和记忆形成可塑性的基因的广泛调节。 摘要：在过去几年中，获取的数据集提供了前所未有且极为细粒度的细节，涉及疼痛感应电路中的基因表达。这似乎很复杂，但基本目标是了解我们如何感觉到疼痛以及在需要时如何控制痛苦。在我们的环境中可能会遇到各种各样的疼痛刺激，并且存在不同的神经元，以感知这些不同类型的疼痛信号。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。我们将使用这些信息来了解疼痛信号以及如何控制疼痛。综上所述，这些数据为疼痛研究界提供了一种变革性的新资源，并将更精确地评​​估实验操作和实验结果的验证。