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：
8280848
负责人：
David D McKemy
金额：
$ 17.89万
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 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8280848
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

DESCRIPTION (provided by applicant): 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 completio 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. PUBLIC HEALTH RELEVANCE: In this application we propose to establish a novel neurogenetic mouse model that will serve as a molecular genetic tool to study the mechanisms of neuronal function and phenotypic identity between distinct subtypes of somatosensory neurons under normal and pathological conditions.

描述（由申请人提供）：传统的神经元基因表达谱分析通常采用某种隔离急性解散的原代神经元的方法，该策略可以在隔离过程中引入细胞不良创伤，并且需要大量样本量以生成足够的起始物质。这些局限性特别关注外周神经系统（PNS）功能上赋予的感觉传递物，因为它们在感觉神经节内代表的细胞类型不当，其基因表达表型对任何形式的扰动都非常敏感。为了克服这些局限性，我们建议使用翻译核糖体亲和力纯化（TRAP）技术来识别遗传靶向体感传入中的翻译mRNA，这种方法尚未在PNS中使用。陷阱涉及标记的核糖体蛋白的表达，因此可以通过免疫亲和力纯化来积极地翻译mRNA。通过靶向特定的细胞群，可以进行基因表达分析，而无需对细胞进行侵入性隔离技术。在这里，我们提出了两个目标，其中将产生转基因小鼠，该目标将靶向模态特异性神经元同类群以在正常条件下进行翻译分析，然后确定该特征在病理条件下如何变化的病理状况如何变化，其特征是疼痛的超敏反应。使用R21机制，我们将瞄准表达TRPM8，冷门控离子通道和体内寒冷温度的主要传感器的感觉神经元的少量子集。 TRPM8-NULL小鼠在各种各样的冷反应中缺乏，从那些被认为凉爽到痛苦的冷反应，并且缺乏损伤引起的感冒过敏。我们假设后一种表型部分是由于该队列中基因表达的改变，正如在一系列病理条件下显示在一般人群中发生的那样，我们将在研究中直接测试这一问题。因此，该探索性建议的完成将在PNS中建立新的分子遗传学方法论，这些方法可用于任何遗传障碍的神经元亚型，从而使基因表达在功能上不同的神经元和亚群中的分子表型评估之间进行了基因表达分析。公共卫生相关性：在本应用中，我们建议建立一种新型的神经遗传小鼠模型，该模型将作为一种分子遗传工具，用于研究正常和病理条件下体感神经元不同亚型之间神经元功能和表型认同的机制。