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，因为它的患病率很高，缺乏 足够的治疗选择。脊柱背角是整合的主要位置 体感信息和基于电路的转换，这些转换是持续疼痛的基础。 我们工作的总体目标是开发针对背喇叭电路的新型疼痛疗法 用于机械性异常。我们使用化学生成受体的细胞类型特异性靶向鉴定 脊柱背角神经元可介导机械性异常性疾病而不会影响急性感觉 在老鼠中。小鼠是这些研究的常用模型系统，因为它们的一般性 与人类相似，遗传操作的便利性和可用的大量工具。 但是，用于治疗目的的脊柱背角神经元需要 了解物种之间的细胞和分子保护。恒河猕猴是 系统发育从与人类更相似，使用重新设计的工具可以用作证明 原理模型系统。为此，我们分析了分子和细胞组织 单细胞转录组学和原位杂交方法的猕猴背角。我们 将这些数据与小鼠和人类数据进行比较以生成一个统一的背喇叭地图集 跨物种的细胞类型。在这里，我们将1）确定人类猕猴的表观遗传景观 和小鼠背角神经元使用单核RNA-和ATAC-SEQ数据以及 机器学习辅助生物信息学方法以识别监管元素（RES） 驱动跨物种的强大细胞类型特异性基因表达。我们将2）筛选候选人Res 用于小鼠的细胞类型特异性，然后使用猕猴，然后使用细胞类型特异性RES 系统地生成跨物种背角的功能图。研究提出 这将生成用于单细胞转录组学并打开的大规模搜索数据库 小鼠，猕猴和人类的染色质区域。重要的是，我们将拥有知识和 病毒载体需要进行猕猴的原则研究新疗法的原则研究证明 机械性异常。