The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 8736696
• 负责人: Michael J. Iadarola
• 金额: $ 74.27万
• 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/8736696
• 关键词: Absence of pain sensation; Acute Pain; Address; Advanced Malignant Neoplasm; Agonist; Analgesics; Animal Model; Animals; Autopsy; Calcium; Canis familiaris; Capsaicin; Cells; Cereals; Clinical Trials; Clonidine; Collaborations; Communities; Computer software; Control Animal; DNA Sequence; Data; Data Analyses; Data Set; Databases; Diphtheria Toxin; Dorsal; Dyes; Environment; Enzymes; Evolution; Excision; Face; Family; Fluorescence; G Protein-Coupled Receptor Genes; Ganglia; Gene Expression; Gene Expression Profile; Generations; Genes; Genetic; Goals; Head; Human; Human Resources; Ibuprofen; Inflammation; Intervention; Intractable Pain; Ion Channel; Knowledge; Label; Leukotriene Receptor; Lidocaine; Lipid Binding; Lipids; Malignant Bone Neoplasm; Malignant Neoplasms; Measurement; Mediating; Messenger RNA; Methods; Microinjections; Modeling; Molecular; Molecular Neurobiology; Monkeys; Morphine; Motor; Mus; National Institute of Mental Health; National Institute on Alcohol Abuse and Alcoholism; Nerve Endings; Neurons; Nociception; Occupations; Opioid; Opioid Receptor; Orphan; Pain; Pain Research; Pain management; Paper; Pathway interactions; Peripheral; Pharmaceutical Preparations; Physiological; Physiology; Population; Preparation; Process; Protocols documentation; RNA; Rattus; Recruitment Activity; Regulation; Reproducibility; Research; Research Infrastructure; Research Methodology; Resiniferatoxin; Resolution; Resources; Rheumatoid Arthritis; Role; Route; Running; Sampling; Scientist; Sensory; Sensory Ganglia; Signal Transduction; Skin; Sorting - Cell Movement; Spinal Cord; Spinal Ganglia; Staging; Stimulus; Structure of trigeminal ganglion; TRPV1 gene; Techniques; Testing; Time; Tissue Banks; Tissue Sample; Tissues; Training; Transcript; Transgenic Organisms; Validation; Work; analog; base; cancer pain; cell killing; cell type; chronic pain; deep sequencing; dorsal horn; drug development; gabapentin; human data; insight; interest; killings; lipid transport; meetings; mu opioid receptors; osteosarcoma; paralogous gene; presynaptic; prototype; receptor; receptor binding; relating to nervous system; research study; species difference; statistics; therapeutic target; transcriptome sequencing; transcriptomics; translational study; transmission process

Overview: Last year we established research methodology and protocols, built an infrastructure of hardware and software, formed collaborative arrangements, trained a team of scientists and support personnel, and performed over 150 sequencing runs on the Illumina HiSeq 2000. Over 15 billion bases of transcriptome sequence information have been generated, consequently, the main effort has been devoted to intensive analysis of the resulting data sets. We have sequenced the transcriptomes of physiologically or genetically labeled pain-sensing neurons sorted by FACS, neurons in dorsal spinal cord during peripheral inflammation and models of rheumatoid arthritis, inflamed peripheral tissue, and axotomized DRG and dorsal and ventral spinal cords. We have sampled multiple time points 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 the pain-sensing neurons and which are in mainly non-pain-sensing neurons such as proprioceptive primary afferents. The ability to form incisive hypotheses regarding pain physiology is greatly advanced by this type of neuron-specific information and we now have quantitative information on all the genes that mediate DRG and sensory and motor spinal cord functions. TRPV1 Transcriptome: One important focus of the neuronal sorting experiments is a particular subpopulation of DRG neurons that express a multifunctional 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. Because of this crucial role, we want to know everything possible about TRPV1-expressing DRG neurons. We isolated TRPV1 neurons by genetic labeling or physiological activation and then performed deep sequencing of the mRNA content using next-gen RNA-Seq. The genetic method expressed a fluorescent marker allowing the TRPV1 DRG neurons to be isolated by FACS. A second strategy was to isolate by pharmacological activation. We loaded primary DRG neurons with a calcium sensitive dye, stimulated them with RTX and sorted the neurons that displayed RTX-induced increases in fluorescence. We also killed the cells either genetically or by microinjection of RTX. Our first paper (in preparation) 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 information on genes expressed by a clinically important population of nociceptive neurons. 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. It has not been clear which paralogs or subunits of drug binding receptors are expressed by different tissues in the pain pathway, yet this becomes clear when expression values for all the relevant genes are obtained quantitatively, at the same time, and with excellent reproducibility between animals and treatments. Additional analgesic targets: The transcriptome experiments also point to new targets for potential analgesic drug development. New targets that are highly differentially expressed in the TRPV1 population include an orphan GPCR, a lipid-binding GPCR, and a leukotriene receptor. In some cases prototype agonists or antagonists are available although their analgesic potential has not been explored. In another example, we observe that the Mu opioid receptor is expressed exclusively in the DRG and not in the dorsal spinal cord. This allows us to conclude that epidural or intrathecal opioid analgesia is solely mediated by a presynaptic action on DRG neurons. Amplification of ongoing studies: The RNA-Seq results also inform and amplify hypothesis-driven studies from our and other groups. In a collaborative work with NIAAA, we observe that certain lipids are TRPV1 agonists. Using the transcriptome databases, we 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 a particular, yet previously unrecognized, family of lipids that may be very important for TRPV1 activation. Canine ganglionic transcriptome: We have nearly completed the canine tissue collection for the cancer pain transcriptome study. The ganglion and spinal cord tissue have been obtained from controls and animals with osteosarcoma that were euthanized because of inadequate pain control or treated with resiniferatoxin and tissues obtained at autopsy. This study was undertaken to test for genes activated by nociceptive input from naturally occurring bone cancer and modulated by treatment. We can also make comparison to parallel studies in mouse, rat and human, although the exact models or cancer problems will be different. This is a unique set of data that will provide new insight into the transcriptomics of cancer pain in a species with a cancer pain problem that is very similar to the human. Cross-species comparison: Another project we are in the process of completing is a species comparison of the trigeminal ganglion transcriptome. The trigeminal ganglia and the medulla (medullary dorsal horn) are equivalent to DRG and spinal cord for the face and head. We have obtained and analyzed these two tissues in collaboration with Dr. Joel Kleinman formerly of NIMH. We have trigeminal ganglion sequence information from mouse, rat, and monkey and soon the dog. Comparisons show several remarkable species differences in degree of expression. This study is providing a new level of cross-species validation of potential therapeutic targets and mechanisms that aid in ascertaining the predictive capability of translational animal models. Summary: The data sets acquired over the past year 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 necessary. 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 trying to figure out 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 will provide a transformative new resource for the pain research community and will allow a new much more precise assessment of experimental manipulations and verification of experimental results.

概述：去年，我们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，培训了一组科学家和支持人员，并在Illumina Hiseq 2000上进行了150多次测序运行。在150亿个基础基础上进行了转录组序列信息的超过150亿个依据，从而使数据集成了依据，从而跨越了依据分析的分析。我们已经测序了由FACS分类的生理或遗传标记的疼痛传感神经元的转录组，外周炎症期间的背脊髓神经元以及类风湿关节炎的模型，发炎的外周组织，以及轴心症状的DRG，DRG，背侧和脊柱脊柱。我们已经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，哪些主要是非吸毒感应神经元中的基因，例如本体感受性的主要传入。通过这种类型的神经元特异性信息，对疼痛生理形成有关疼痛生理的敏锐假设的能力大大提高了，我们现在拥有有关介导DRG，感觉和运动脊髓功能的所有基因的定量信息。 TRPV1转录组：神经元分选实验的一个重要重点是对DRG神经元的特殊亚群，表达了称为TRPV1的多功能热化学pH-和脂质响应离子通道。 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。由于这一至关重要的角色，我们想了解有关表达TRPV1的DRG神经元的所有可能。 我们通过遗传标记或生理激活分离了TRPV1神经元，然后使用下一代RNA-Seq对mRNA含量进行了深层测序。遗传方法表达了荧光标记物，允许FACS分离TRPV1 DRG神经元。第二种策略是通过药理激活分离。 我们用钙敏感的染料加载了原代DRG神经元，用RTX刺激了它们，并对显示RTX诱导的荧光增加的神经元进行了排序。我们还通过遗传或微分注射RTX杀死了细胞。我们的第一篇论文（准备）概述了转录组是由遗传标记的TRPV1神经元和神经节引起的，其中TRPV1神经元通过表达白喉毒素或对RTX的显微注射而被删除。 这为临床上重要的伤害性神经元人群表达的基因提供了全面的新信息。 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。 目前尚不清楚在疼痛途径中，不同组织表达了哪些旁系同源物或亚基，但是当所有相关基因的表达值同时获得，并且动物和治疗之间的可重复性极好时，这很清楚。 其他镇痛靶标：转录组实验还指出了潜在镇痛药物发育的新靶标。 在TRPV1种群中高度差异表达的新靶标包括孤儿GPCR，脂质结合GPCR和白细胞受体。 在某些情况下，尽管尚未探索其镇痛势，但仍有原型激动剂或拮抗剂可用。 在另一个例子中，我们观察到MU阿片受体仅在DRG中而不是在背脊髓中表达。这使我们得出结论，硬膜外或鞘内阿片类镇痛仅由突触前作用对DRG神经元的作用介导。 正在进行的研究的放大：RNA-seq结果还为我们和其他群体的假设驱动的研究提供了信息。在与NIAAA的合作工作中，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，生成，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但未识别的脂质家族的见解，这对于TRPV1激活可能非常重要。 犬神经节转录组：我们几乎完成了用于癌症疼痛转录组研究的犬组织收集。 神经节和脊髓组织是从具有骨肉瘤的对照和动物中获得的，这些对照和动物因疼痛控制不足而被安乐死，或用尸检时获得的树脂毒素和组织治疗。这项研究是为了测试来自天然发生的骨癌的伤害感受输入并通过治疗调节而激活的基因。尽管确切的模型或癌症问题不同，但我们还可以与小鼠，大鼠和人类的平行研究进行比较。 这是一组独特的数据，它将为患有癌症疼痛问题的癌症疼痛的转录组学提供新的见解，与人类非常相似。 跨物种比较：我们正在完成的另一个项目是三叉神经节转录组的物种比较。三叉神经节和髓质（髓质背角）等同于DRG和脊髓的脸部和头部。 我们已经与NIMH的乔尔·克莱曼（Joel Kleinman）博士合作获得并分析了这两个组织。 我们有来自小鼠，大鼠和猴子的三叉神经节序列信息以及狗很快。比较显示表达程度的几种显着物种差异。这项研究为潜在的治疗靶标和机制提供了新的跨物种验证，有助于确定转化动物模型的预测能力。 摘要：过去一年中获得的数据集为疼痛传感电路中的基因表达提供了前所未有且极为细的细节。这似乎很复杂，但基本目标是了解我们如何感觉到痛苦以及在必要时如何控制痛苦。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。我们正在尝试确切弄清楚哪些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，这些数据将为疼痛研究界提供变革性的新资源，并将允许对实验操作的新新评估和实验结果的验证。