Translational profiling of somatosensory afferent neurons

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一些分离急性解离的原代神经元的方法，这种策略可能在分离过程中对细胞引入不希望的创伤，并且需要大的样本量以产生足够的起始材料。这些限制是特别关注的功能不同的感觉传入神经在周围神经系统（PNS），因为它们是代表性差的感觉神经节内的细胞类型，其基因表达表型是非常敏感的任何形式的扰动。为了克服这些局限性，我们建议使用翻译核糖体亲和纯化（TRAP）技术来鉴定遗传靶向体感传入神经中的翻译mRNA，这种方法尚未在PNS中使用。TRAP涉及标记的核糖体蛋白的表达，使得可以通过免疫亲和纯化来分离主动翻译的mRNA。通过靶向特定的细胞群体，可以进行基因表达谱分析，而无需使细胞经受侵入性分离技术。在这里，我们提出了两个目的，其中将产生转基因小鼠，其靶向模态特异性神经元组群以在正常条件下进行翻译谱分析，然后确定该谱在以疼痛性超敏反应为特征的病理条件下如何变化。使用R21机制，我们将靶向表达TRPM8的感觉神经元的一小部分，TRPM8是一种冷门控离子通道，也是体内低温的主要传感器。TRPM8基因缺失的小鼠缺乏广泛的冷反应，从感觉凉爽到疼痛的冷，并且缺乏损伤诱导的冷超敏反应。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，正如已显示在一系列病理条件下发生在一般人群中一样，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在PNS中建立新的分子遗传学方法，其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亚群内的分子表型评估。 公共卫生相关性：在本申请中，我们建议建立一种新的神经遗传小鼠模型，将作为一种分子遗传学工具，以研究正常和病理条件下不同亚型的体感神经元之间的神经元功能和表型同一性的机制。

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.