Functional and genetic characterization of human DRG and spinal cord at single cell resolution

单细胞分辨率下人类 DRG 和脊髓的功能和遗传特征

• 批准号: 10707419
• 负责人: Robert W Gereau
• 金额: $ 48.83万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10707419
• 关键词: Address; American; Analgesics; Anatomy; Axon; Biological Assay; Catalogs; Cells; Central Nervous System; Data; Data Set; Development; Diagnosis; Electrophysiology (science); Gene Expression Profile; Genetic; Genetic Transcription; Genetic study; Goals; Helping to End Addiction Long-term; Human; Knowledge; Location; Maintenance; Maps; Membrane; Molecular; Morphology; Nervous System; Neurons; Opioid; Pain; Pain management; Patients; Pharmaceutical Preparations; Phenotype; Physiological; Physiology; Population; Positioning Attribute; Posterior Horn Cells; Preparation; Property; Protocols documentation; Recording of previous events; Research; Resolution; Resources; Risk; Slice; Spinal; Spinal Cord; Spinal Ganglia; Structure; Synapses; Tissues; United States National Institutes of Health; Vertebral column; Work; addiction; aspirate; cell type; chronic pain; chronic pain patient; chronic painful condition; evidence base; novel; opioid epidemic; opioid misuse; opioid mortality; opioid use; opioid use disorder; patch clamp; patch sequencing; reconstruction; single-cell RNA sequencing; substance misuse; substance use; tool; transcriptome sequencing; transcriptomics; transmission process

Specific Aims: Tens of millions of Americans suffer from chronic pain. Unfortunately for these patients, there is very little we can offer in the way of treatments. Opioids represent the main tool for treating pain, but their use in chronic pain conditions suffers from a poor evidence base and the inherent risk of addiction. The current crisis of opioid-related deaths highlights the risk associated with widespread opioid use. The parallel crises of chronic pain and opioid-related deaths has led the NIH to launch the HEAL Initiative, with one of the major goals being the identification of novel analgesics for the treatment of pain, with a focus on drugs that are non-addicting. The PRECISION Human Pain Network seeks to provide foundational data on the diversity among cell types that comprise the pain neuraxis. Goals for our proposed PRECISION Human Pain Network U19 center include the development of optimized experimental protocols and functional assays utilizing primary human cells and tissue (including dorsal root ganglion neurons and spinal cord slices), and the elucidation of comprehensive molecular/cellular phenotypes that underlie human pain transduction, transmission, and processing under different pain conditions. This project seeks to increase our understanding of the physiological properties of human neurons in the pain neuraxis. The three aims here will begin to catalog the functional profiles and transcriptinoal signatures of DRG and spinal neurons from donors with and without a history of pain or opioid use. We will continue our work to optimize protocols for cutting edge functional and genetic studies in human nervous system tissue. We will work with other centers to optimize protocols to assess physiological, transcriptional, and morphological features of human DRG and spinal cord neurons at cellular resolution, and how these properties are impacted in tissues recovered from patients with a history of pain. Because our donor population also includes patients with substance use and misuse, and in some cases this overlaps with chronic pain conditions, we will also characterize similar properties of DRG and spinal cord neurons in patients with and without a recent history of opioid use. These studies will provide foundational knowledge of human neurons in the pain neuraxis by mapping electrophysiological features of neurons in the human DRG and spinal cord onto genetically-defined subpopulations of human DRG or morphological classes of spinal neurons. The study will also profile tissues derived from donors with and without a history of chronic pain or opioid use/misuse, providing opportunity for new understanding of the impact of pain and substance use on these neurons in the human pain neuraxis. Another major deliverable from the present study will be the development of optimized protocols for key aspects of the proposed analyses, including protocols for RNA sequencing from patch clamped neurons (Patch-seq) and for the preparation, maintenance, and recording form human spinal cord slices.

具体目标：数千万美国人患有慢性疼痛。对于这些患者来说， 我们能提供的治疗方法非常少。阿片类药物是治疗疼痛的主要工具， 在慢性疼痛的条件下，患有不良的证据基础和成瘾的固有风险。当前危机 与类阿片有关的死亡率的下降突出了与广泛使用类阿片有关的风险。慢性病的平行危机 疼痛和阿片类药物相关的死亡导致NIH启动了HEAL倡议，其主要目标之一是 确定用于治疗疼痛的新型镇痛药，重点是非成瘾性药物。的 PRECISION人类疼痛网络旨在提供有关细胞类型多样性的基础数据， 包括疼痛神经轴。我们提议的PRECISION人类疼痛网络U19中心的目标包括 利用原代人类细胞和组织开发优化的实验方案和功能测定 （包括背根神经节神经元和脊髓切片），并阐明了全面的 分子/细胞表型是人类疼痛转导、传递和加工的基础， 不同的疼痛状况。该项目旨在增加我们对生理特性的理解， 疼痛神经轴中的人类神经元。这里的三个目标将开始对功能概要进行分类， 来自有和没有疼痛或阿片类药物史的供体的DRG和脊髓神经元的转录特征 使用.我们将继续我们的工作，以优化方案的尖端功能和遗传研究在人类 神经系统组织我们将与其他中心合作，优化方案，以评估生理， 人DRG和脊髓神经元在细胞分辨率下的转录和形态学特征，以及 这些特性如何在从有疼痛史的患者身上回收的组织中受到影响。因为我们的捐赠者 人群还包括物质使用和滥用的患者，在某些情况下，这与慢性疾病重叠。 疼痛条件下，我们还将表征DRG和脊髓神经元的相似特性， 没有近期阿片类药物使用史这些研究将提供人类神经元的基础知识， 通过将人DRG和脊髓中神经元的电生理特征映射到 人DRG的遗传学定义的亚群或脊髓神经元的形态学类别。这项研究将 还分析了来自有和没有慢性疼痛或阿片类药物使用/滥用史的供体的组织， 有机会重新理解疼痛和物质使用对人类疼痛中这些神经元的影响 神经轴本研究的另一个主要成果是开发优化的方案， 提出的分析的关键方面，包括从膜片钳神经元RNA测序的协议 （Patch-seq）和用于制备、维持和记录人脊髓切片。