Cell-type specific molecular and functional analyses to target dorsal horn pain circuitry in mice and non-human primates

针对小鼠和非人类灵长类动物背角疼痛回路的细胞类型特异性分子和功能分析

• 批准号: 10863324
• 负责人: Andreas Robert Pfenning
• 金额: $ 49.91万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-08 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10863324
• 关键词: ATAC-seq; Acute; Affect; Afferent Neurons; Atlases; Automobile Driving; Bioinformatics; Biological Models; Cell Nucleus; Cell model; Cells; Cholecystokinin; Chromatin; Chronic; Clinical; Communities; Computer Analysis; Data; Data Set; Engineering; Epigenetic Process; Esthesia; Future; Gene Expression; Genes; Genetic; Goals; Hand; High Prevalence; Horns; Human; In Situ Hybridization; Individual; Inflammatory; Injury; Knowledge; Laboratories; Ligands; Macaca; Macaca mulatta; Machine Learning; Maps; Measures; Mediating; Methods; Modeling; Molecular; Movement; Mus; Nerve; Neurons; Neuropathy; Pain; Pain management; Parvalbumins; Patients; Persistent pain; Phylogenetic Analysis; Population; Positioning Attribute; Posterior Horn Cells; Process; Regulatory Element; Research; Site; Specificity; Spinal; Technology; Therapeutic; Time; Tissues; Touch sensation; Transgenic Mice; Translating; Vertebral column; Viral Vector; Work; behavior test; candidate identification; cell type; chronic pain patient; design; dorsal horn; excitatory neuron; experimental study; genetic manipulation; human data; improved; inhibitory neuron; insight; integration site; machine learning algorithm; mechanical allodynia; neurotransmission; nonhuman primate; novel; novel therapeutics; pain patient; protein kinase C gamma; receptor; recombinase; screening; searchable database; single cell sequencing; single nucleus RNA-sequencing; somatosensory; targeted treatment; therapeutic candidate; therapeutic target; tool; transcriptomics; virus genetics

Project Summary Persistent pain remains a major clinical problem because of its high prevalence and the lack of adequate treatment options. The spinal dorsal horn is a major site for the integration of somatosensory information and for circuit-based transformations that underlie persistent pain. The overall goal our work is to develop novel pain therapies that target the dorsal horn circuitry for mechanical allodynia. We use cell type-specific targeting of chemogenic receptors to identify spinal dorsal horn neurons that mediate mechanical allodynia without affecting acute sensation in mice. Mice are a commonly used model system for these studies because of their general similarity to humans, the ease of genetic manipulations and the large number of tools available. However, targeting spinal dorsal horn neurons for therapeutic purposes requires an understanding of the cellular and molecular conservation between species. Rhesus macaque are phylogenetically more similar to humans and with re-engineered tools can serve as a proof-of- principle model system. To this end, we analyzed the molecular and cellular organization of the macaque dorsal horn by single cell transcriptomics and in situ hybridization methods. We compared these data to mouse and human data to generate a harmonized atlas of dorsal horn cell types across species. Here we will 1) Identify the epigenetic landscape of human, macaque and mouse dorsal horn neurons using single nucleus RNA- and ATAC-Seq data together with machine learning-assisted bioinformatic approaches to identify regulatory elements (REs) that drive robust cell type-specific gene expression across species. We will 2) screen candidate REs for cell-type specificity in mice and subsequently macaque and then use the cell type-specific REs to systematically generate a functional map of the dorsal horn across species. Studies proposed here will generate large-scale searchable databases for single cell transcriptomics and open chromatin regions in mice, macaque and human. Importantly, we will have the knowledge and viral vectors needed to pursue macaque proof-of-principle studies of novel therapies for mechanical allodynia.

项目摘要 持续性疼痛仍然是一个主要的临床问题，因为它的高患病率和缺乏治疗。 充足的治疗选择。脊髓背角是整合的主要部位， 躯体感觉信息和基于电路的转换，构成持续性疼痛的基础。 我们工作的总体目标是开发针对背角回路的新型疼痛疗法 治疗机械性异常性疼痛我们使用细胞类型特异性靶向化学受体， 脊髓背角神经元介导机械性异常性疼痛而不影响急性感觉 对小鼠小鼠是这些研究中常用的模型系统，因为它们的一般特性， 与人类的相似性，基因操作的容易性和大量可用的工具。 然而，靶向脊髓背角神经元用于治疗目的需要一种方法， 了解物种之间的细胞和分子保守性。恒河猴是 在遗传学上与人类更相似，并使用重新设计的工具可以作为证据- 原理模型系统为此，我们分析了细胞的分子和细胞组织， 用单细胞转录组学和原位杂交方法研究猕猴背角的细胞凋亡。我们 将这些数据与小鼠和人类数据进行比较，以生成协调的背角图谱 不同种类的细胞。在这里，我们将1）确定人类，猕猴， 和小鼠背角神经元使用单核RNA-和ATAC-Seq数据连同 机器学习辅助的生物信息学方法来识别调控元件（RE）， 驱动跨物种的强大细胞类型特异性基因表达。我们将2）筛选候选RE 用于小鼠和随后的猕猴中的细胞类型特异性，然后使用细胞类型特异性RE 来系统地绘制出不同物种的背角的功能图。拟议的研究 这里将为单细胞转录组学产生大规模可搜索数据库， 小鼠、猕猴和人类的染色质区域。重要的是，我们将拥有知识， 病毒载体需要进行猕猴原理验证研究的新疗法 机械性异常性疼痛