Translational profiling of somatosensory afferent neurons

摘要 传统的神经元基因表达谱通常采用某种方法来分离急性分离的 初级神经元，这是一种在分离过程中可能给细胞带来不良创伤的策略， 并且需要较大的样本量以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 感觉神经节内缺乏代表性的细胞类型，其基因表达表型极其敏感 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)技术识别基因靶向的躯体感觉传入中翻译的mRNAs， 这是一种尚未在PNS中使用的方法。TRAP涉及标记核糖体蛋白的表达 这样就可以通过免疫亲和纯化来分离主动翻译的mRNAs。通过瞄准特定的细胞 在人群中，基因表达谱可以在不使细胞受到侵入性分离的情况下进行 技巧。在这里，我们提出了两个目标，在这个目标中，将产生针对一种模式的转基因小鼠- 在正常条件下翻译简档的特定神经元队列，然后确定如何 这一特征在以疼痛过敏为特征的病理条件下发生变化。使用R21 机制，我们将针对表达TRPM8的一小部分感觉神经元，TRPM8是一种冷门离子通道 以及体内寒冷温度的主要传感器。TRPM8基因缺失的小鼠在一系列感冒中都存在缺陷 反应，从那些被认为令人愉快的凉爽到痛苦的寒冷，以及缺乏受伤导致的寒冷 过敏症。我们推测，后一种表型的部分原因是基因表达的改变 队列，正如已经证明在一系列病理条件下在普通人群中发生的那样，一种观点 我们将在我们的学习中直接测试。因此，这一探索性提案的完成将确立小说 PNS中的分子遗传学方法可以用于任何遗传上易处理的神经元亚型， 允许在功能不同的神经元之间进行基因表达谱分析和分子评估 亚群内的表型。

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.