The Pain Neural Transcriptome

疼痛神经转录组

• 批准号: 9555581
• 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/9555581
• 关键词: Absence of pain sensation; Acute Pain; Address; Advanced Malignant Neoplasm; Affect; Agonist; Analgesics; Anesthesia procedures; Anesthetics; Animal Model; Animals; Autopsy; Axon; Behavioral; Biological Models; Brain region; Breathing; Calcium; Cancer Control; Cancer Patient; Canis familiaris; Capsaicin; Capsicum; Cells; Cereals; Cerebral cortex; Clinical; Clinical Trials; Clonidine; Communication; Communities; Computer software; Copy Number Polymorphism; Data; Data Set; Databases; Defect; Diphtheria Toxin; Dorsal; Environment; Enzymes; Epidemic; Evolution; Fluorescence; G-Protein-Coupled Receptors; Ganglia; Gene Expression; Gene Expression Profile; General Anesthesia; Generations; Genes; Genetic Techniques; Genetic Variation; Goals; Headache; Hippocampus (Brain); Human; Human Genetics; Human Resources; Ibuprofen; Inflammation; Inhalation Anesthetics; Intervention; Investigation; Ion Channel; Knowledge; Label; Laboratories; Lead; Lidocaine; Ligands; Lipids; Mass Spectrum Analysis; Measures; Mediating; Memory; Messenger RNA; Metabolic Pathway; Methodology; Modeling; Molecular; Molecular Biology; Molecular Neurobiology; Morphine; Motor; Mus; Myelin Sheath; Nerve Endings; Neuroglia; Neurons; Nociception; Nociceptors; Nuclear; Occupations; Opioid; Orphan; Pain; Pain Research; Pain management; Paper; Pathologic; Pathway interactions; Patients; Peripheral; Peripheral Nerves; Peripheral Nervous System; Persistent pain; Pharmacology; Physiological; Physiology; Population; Preparation; Process; Production; Property; Proteins; Protocols documentation; Pruritus; Psoriasis; Publications; Publishing; Rattus; Recovery; Recruitment Activity; Regulation; Reporting; Research; Research Infrastructure; Research Methodology; Resiniferatoxin; Resistance; Resolution; Resources; Rheumatoid Arthritis; Rodent Model; Role; Running; Sampling; Schwann Cells; Scientist; Sensory; Sensory Ganglia; Short-Term Memory; Sodium Channel; Spinal Cord; Spinal Ganglia; Supporting Cell; Synapses; TRPV1 gene; Techniques; Time; Tissue Sample; Tissues; Training; Transcription Process; Transcriptional Regulation; Transgenic Organisms; Vulnerable Populations; Work; aged; analog; cancer pain; chronic pain; cognitive function; cohort; comparative; deep sequencing; drug development; executive function; experimental study; functional plasticity; gabapentin; human tissue; insight; killings; mouse model; novel strategies; paralogous gene; peripheral pain; receptor; relating to nervous system; statistics; time use; transcriptome; transcriptome sequencing; transcriptomics; translational study; 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. We have performed hundreds of deep sequencing runs in various species and models and have obtained over 50 billion 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 rheumatoid arthritis, inflamed peripheral tissue, axotomized DRG, dorsal and ventral spinal cords and peripheral nerve. We are also investigating transcriptional processes affected by general anesthesia in higher order brain regions. 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 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, 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. 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 sequencing RNA-Seq on various animal and human ganglionic preparations targeting TRPV1 neurons. We published a report in mouse and rats using genetic techniques to FACS isolate TRPV1+ DRG neurons and to obtain the inverse population by killing the TRPV1+ neurons with diphtheria toxin or RTX treatment. and isolating TRPV1+ neurons by agonist-activated calcium fluorescence. Our initial publication outlines the transcriptome results from several of the above manipulations and provides a comprehensive transcriptomic profile of this clinically important population of nociceptive neurons. We followed this up in another publication in which we distinguished the contribution of Schwann cells versus neurons to the DRG transcriptome. A third publication has been submitted in which we isolated TRPV1+ neurons by agonist-activated calcium fluorescence and sequenced DRGs obtained at autopsy from one of our human cancer pain patients who had been treated with RTX and a cohort of canines with cancer pain who had been treated for pain with RTX. Interestingly these data demonstrated that the most sensitive neuronal component is the centrally projecting axons that contain RTX and the cell bodies in the ganglion 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. 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 receptors are expressed by neurons in the pain pathway. Our data show that when expression for all the relevant genes are obtained quantitatively a 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. We published a report that began with transcriptomic profiling of lipid generating genes to define a metabolic pathways for production of potentially neuroactive lipids. This novel approach predicted two new lipids which we identified 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 in a rodent model of the hemideletion. In these studies, transcriptomic profiling allows for more precise hypothesis generation. Anesthesia Transcriptome: We are in the process of completing an assessment of the effects of inhalation general anesthesia on cortical and hippocampal transcriptomes and associated proteins identified from the gene analysis. This is the initial step to a larger study on general anesthesia and cognitive function in aged animals. In humans, general anesthesia can be deleterious to cognitive function and 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 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急性和慢性疼痛模型以及人类患者或验尸样本中转录组水平上的疼痛传感神经元和周围组织的分子生物学。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架构，形成了协作安排，培训了一个科学家团队，并支持人员使用RNA-Seq的方法。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，并获得了超过500亿个转录组序列信息的读取。 我们在周围炎症期间在生理或遗传标记的疼痛传感神经元，背侧脊髓中神经元的数据集的分析中参与了大量参与，在外周炎症，类风湿关节炎的模型，发炎的外围性DRG，轴向dRG，脊髓索和脊柱脊髓和外侧脊髓和外围神经。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。 对多个时间点进行采样，以遵循干预措施的演变和分辨率，每个点在每个点上都有足够的样本以允许统计比较。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在疼痛感应神经元中，哪些基因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，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。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神经元的各种动物和人类神经节制剂进行了深入测序RNA-Seq。我们使用遗传技术在FACS分离TRPV1+ DRG神经元中发表了一份小鼠和大鼠的报告，并通过用白喉毒素或RTX治疗杀死TRPV1+神经元来获得逆种群。 并通过激动剂激活的钙荧光分离TRPV1+神经元。我们的首次出版物概述了上述几个操作的转录组引起的，并提供了临床上重要的伤害性神经元种群的全面转录组概况。 我们在另一份出版物中遵循了这一点，在该出版物中，我们区分了Schwann细胞与神经元对DRG转录组的贡献。 已经提交了第三份出版物，我们通过激动剂激活的钙荧光分离了TRPV1+神经元，并从尸检时从我们的一名人类癌症疼痛患者中获得的DRG进行了测序，这些患者接受了RTX治疗，并接受了RTX治疗的一系列犬类，患有RTX治疗的癌症疼痛。有趣的是，这些数据表明，最敏感的神经元成分是包含RTX的中心突出轴突，而神经节中的细胞体对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。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，尚不清楚疼痛途径中神经元表达哪些分子旁系同源物或受体。我们的数据表明，当对所有相关基因的表达进行定量获得时，就会出现更多信息图。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。我们发表了一份报告，该报告始于脂质产生基因的转录组分析，以定义生产潜在神经活性脂质的代谢途径。这种新颖的方法预测了我们使用质谱法确定的两种新脂质。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，我们能够评估在啮齿动物的啮齿动物模型中，在感觉神经节和脊髓中神经元的完整性。 在这些研究中，转录组分析允许产生更精确的假设。 麻醉转录组：我们正在完成对吸入大麻醉对皮质和海马转录组以及从基因分析中鉴定的相关蛋白质的影响的评估。这是对衰老动物的全身麻醉和认知功能进行更大研究的第一步。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麻醉和恢复能力引起的分子水平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：过去几年中获得的数据集为疼痛感应电路和麻醉敏感大脑区域中的基因表达提供了前所未有且极为细粒度的细节。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。