Translational profiling of somatosensory afferent neurons

体感传入神经元的翻译分析

基本信息

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
项目类别：
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

项目摘要

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通常采用一些分离急性解离的方法原代神经元，一种可能在分离过程中给细胞带来不良创伤的策略，并且需要大样本量才能产生足够的起始材料。这些限制是特别关注周围神经系统（PNS）中功能不同的感觉传入感觉神经节内代表性较差的细胞类型，其基因表达表型非常敏感任何形式的扰动。为了克服这些限制，我们建议使用翻译核糖体亲和力纯化 (TRAP) 技术，用于识别基因靶向体感传入中的翻译 mRNA，这种方法尚未在 PNS 中使用。 TRAP 涉及标记核糖体蛋白的表达这样可以通过免疫亲和纯化来分离主动翻译的mRNA。通过靶向特定细胞群体中，无需对细胞进行侵入性分离即可进行基因表达谱分析技术。在这里，我们提出了两个目标，其中将产生针对某种模式的转基因小鼠—— 在正常条件下进行翻译分析的特定神经元队列，然后确定如何这种情况在以痛苦的超敏反应为特征的病理条件下会发生变化。使用R21 机制，我们将针对表达TRPM8（一种冷门控离子通道）的一小部分感觉神经元和体内低温的主要传感器。 TRPM8 缺失小鼠缺乏多种感冒功能反应，从那些被认为凉爽宜人到令人痛苦的寒冷，并且没有受伤引起的寒冷超敏反应。我们假设后一种表型部分是由于该基因表达的改变所致队列，正如已被证明在一系列病理条件下发生在一般人群中的那样，一个假设我们将直接在我们的学习中进行测试。因此，这项探索性提案的完成将建立新的 PNS 中的分子遗传学方法可用于任何遗传上易处理的神经元亚型，允许功能不同的神经元之间的基因表达谱分析和分子评估亚群体内的表型。