Translational profiling of somatosensory afferent neurons

体感传入神经元的翻译分析

• 批准号: 8413609
• 负责人: David D McKemy
• 金额: $ 20.77万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8413609
• 关键词: Afferent Neurons; Affinity Chromatography; Cell Separation; Cells; Chronic; Detection; Development; Exploratory/Developmental Grant; Fluorescence-Activated Cell Sorting; Functional disorder; Gated Ion Channel; Gene Expression; Gene Expression Profile; Gene Expression Profiling; General Population; Genes; Genetic Markers; Heterogeneity; Hypersensitivity; Inflammatory; Injury; Knockout Mice; Mediating; Messenger RNA; Methodology; Methods; Modality; Modification; Molecular; Molecular Genetics; Mus; Nature; Neuraxis; Neurons; Nociceptive Stimulus; Pain; Pathology; Peripheral Nervous System; Phenotype; Play; Population; Procedures; Ribosomal Proteins; Ribosomes; Role; Sample Size; Sensory; Sensory Ganglia; Stimulus; System; Techniques; Temperature; Testing; Therapeutic; Touch sensation; Transgenic Mice; Transgenic Organisms; Translating; Trauma; Validation; Variant; cell type; chronic pain; clinically relevant; cohort; cold temperature; effective therapy; emergency service responder; in vivo; molecular phenotype; mouse model; neurogenetics; novel; prevent; promoter; receptor; response; sensor; sensory mechanism; somatosensory; tool; transgene expression

Summary Traditional neuronal gene expression profiling normally employs some method of isolating acutely dissociated primary neurons, a strategy that can introduce undesirable trauma to the cell during the isolation procedures, as well as requires a large sample size in order to generate sufficient starting material. These limitations are of particular concern for functionally-distinct sensory afferents in the peripheral nervous system (PNS) as they are poorly represented cell-types within sensory ganglia whose gene expression phenotype is exquisitely sensitive to any form of perturbation. To overcome these limitations we propose to use the translating ribosome affinity purification (TRAP) technique to identify translating mRNAs in genetically targeted somatosensory afferents, an approach that has yet to be used in the PNS. TRAP involves the expression of a tagged ribosomal protein such that actively translating mRNAs can be isolated by immunoaffinity purification. By targeting specific cell populations, gene expression profiling can be performed without subjecting cells to invasive isolation techniques. Here we propose two Aims in which transgenic mice will be generated that target a modality- specific neuronal cohort for translational profiling under normal conditions, followed by a determination of how this profile changes under pathological conditions characterized by painful hypersensitivity. Using the R21 mechanism, we will target the small subset of sensory neurons that express TRPM8, a cold-gated ion channel and the principal sensor of cold temperatures in vivo. TRPM8-null mice are deficient in a wide array of cold responses, from those perceived as pleasantly cool to painfully cold, and lack injury-induced cold hypersensitivity. We hypothesize that this latter phenotype is partly due to altered gene expression within this cohort, as has been shown to occur in the general population under a range of pathological conditions, a posit we will directly test in our studies. Thus, the completion of this exploratory proposal will establish novel molecular genetic methodologies in the PNS that can be used in any genetically tractable neuronal subtype, allowing gene expression profiling between functionally distinct neurons and assessment of molecular phenotypes within sub-populations.

概括 传统的神经元基因表达谱分析通常采用某种方法来隔离急性解离 原发性神经元，这种策略可以在隔离程序中引入细胞不良创伤， 以及需要大型样本量以生成足够的起始材料。这些限制是 特别关注外周神经系统（PN）中具有功能赋予的感觉传入 感觉神经节内的细胞类型不良的基因表达表型非常敏感 到任何形式的扰动。为了克服这些限制，我们建议使用翻译核糖体亲和力 纯化（陷阱）技术以识别遗传靶向体感传入中的翻译mRNA， PNS尚未使用的方法。陷阱涉及标记的核糖体蛋白的表达 这样，可以通过免疫亲和力纯化来分离积极翻译mRNA。通过靶向特定细胞 种群，可以进行基因表达分析，而无需对细胞进行侵入性分离 技术。在这里，我们提出了两个目标，其中将产生转基因小鼠，以靶向模态 - 特定的神经元队列在正常条件下进行翻译分析，然后确定如何 该特征在病理状况下发生变化，其特征是疼痛性超敏反应。使用R21 机制，我们将针对表达TRPM8的感官神经元的小子集，一种冷门控离子通道 以及体内寒冷温度的主要传感器。 TRPM8-NULL小鼠在各种各样的寒冷中缺乏 回答，从那些被认为愉快的凉爽到痛苦的冷，缺乏伤害引起的寒冷的反应 高敏性。我们假设后一种表型部分是由于基因表达改变了 队列，正如在一系列病理条件下显示在一般人群中发生的那样， 我们将在我们的研究中直接测试。因此，该探索性建议的完成将建立新颖 PNS中的分子遗传学方法论可用于任何遗传障碍的神经元亚型， 允许在功能不同的神经元和分子评估之间进行基因表达分析 亚种群中的表型。