Epigenetic regulation in neuropathic pain

描述（由申请人提供）：现在有相当多的证据表明，神经损伤后从急性疼痛到慢性疼痛的转变反映了神经系统的不适应可塑性，这在生理、结构、生化和分子水平上都是明显的。其结果是对通常无害的刺激（异常性疼痛）有持续的疼痛反应，对正常疼痛刺激（痛觉过敏）有夸大的疼痛反应。许多这些变化是基因表达持续改变的产物和/或原因。例如，先前的微阵列研究和我们初步的RNA-seq分析发现，在背根神经节（DRG）中，神经损伤诱导钙通道alpaha2delta1亚基（Cacnalpaha2delta1）和ATF3转录因子上调，而最常用的神经性疼痛药物加巴喷丁的靶点是钙通道alpaha2delta1亚基。有趣的是，我们对Cacnalpaha2delta1启动子的初步分析表明，它含有一个ATF结合位点。由于ATF家族的成员结合CBP的KIX结构域，使基因启动子上的组蛋白乙酰化并调节基因转录，我们验证了CBP通过表观遗传调节DRG中的基因表达来促进周围神经损伤的神经性疼痛后果的假设。事实上，在CBP KIX结构域发生突变的小鼠中，CBP与转录因子（包括ATF）之间的结合大大减少，我们发现神经损伤引起的机械超敏反应（神经性疼痛的关键行为）是非常短暂的。我们随后的初步RNA-Seq分析确定了DRG中的38个基因，这些基因在CBP突变小鼠中神经损伤诱导的上调显着降低。这些基因包括cacnalpaha2delta1和其他一些先前与神经损伤有关的基因（如NPY、甘丙肽），但重要的是，许多基因尚未与持续性疼痛有关，也未在先前的微阵列研究中发现。我们提出的研究将验证CBP对这些基因的调节确实对神经性疼痛的持续至关重要的假设。在Specific Aim 1中，我们将通过定量RT-PCR、Western blot、免疫组织化学和原位杂交，在神经性疼痛模型中验证我们的RNA-Seq结果。这些研究将确定表达这些cbp调节基因的DRG神经元（或胶质/卫星细胞）亚群。在Specific Aim 2中，我们将通过研究CBP的启动子结合以及CBP特异性组蛋白在cnalpaha2delta1和其他CBP相关基因启动子上的乙酰化，来检测CBP在神经性疼痛模型中调控这些基因的功能。最后，在Specific Aim 3中，我们将研究药物抑制CBP KIX结构域后的神经性疼痛行为。我们还将在神经性疼痛模型中研究KIX抑制对DRG中cbp相关基因表达和表观遗传调控的影响。总之，这些研究将剖析慢性神经性疼痛的表观遗传学景观，并确定其管理的潜在目标。我之前的研究训练与博士。神经科学和分子生物学的Eric Kandel和Howard Nash为我提供了设计、执行和分析实验结果的技能和知识。然而，我的长期职业目标是成为一名内科科学家和一名独立的研究者，研究慢性疼痛的机制，特别是表观遗传调控在慢性神经性疼痛的发生和恢复中的作用。有了这个目标，有三个重要的领域，我需要额外的培训，指导和经验：(1)在疼痛研究中使用的传统技术和模型，(2)尖端的分子/表观遗传学技术，(3)高通量测序数据的计算分析。除了获得实践经验，我还计划在冷泉港和加州大学伯克利分校学习表观遗传学和计算数据分析课程。在这份申请中，我提出了一份详细的职业发展计划，这将使我能够获得额外的培训和指导研究经验，以实现这些目标，并成功地竞争R01资金，从而实现作为首席研究员的独立性。我的部门保证有实验室空间和80%的专业时间用于我的研究，这两项都不取决于我是否获得了这个职业奖。

DESCRIPTION (provided by applicant): There is now considerable evidence that the transition from acute to chronic pain following nerve injury reflects a maladaptive plasticity of te nervous system that is manifest at physiological, structural, biochemical and molecular levels. The result is a condition in which there is ongoing pain in response to normally innocuous stimuli (allodynia) and exaggerated pain in response to normally painful stimuli (hyperalgesia). Many of these changes are the product and/or the cause of lasting alterations in gene expression. For example, previous microarray studies and our preliminary RNA-seq analysis found that in dorsal root ganglion (DRG), nerve injury induces up regulation of the calcium channel alpaha2delta1 subunit (Cacnalpaha2delta1), which is targeted by the most commonly used neuropathic pain medication gabapentin, and the ATF3 transcription factor. Interestingly, our preliminary analysis of the Cacnalpaha2delta1 promoter showed that it contains an ATF binding site. As members of the ATF family bind the KIX domain of CBP, which acetylates histones at gene promoters and regulates gene transcription, we tested the hypothesis that CBP contributes to the neuropathic pain consequences of peripheral nerve injury by epigenetically regulating gene expression in the DRG. Indeed, in mice in which the CBP KIX domain is mutated so that the binding between CBP and transcription factors, including ATF, is greatly reduced, we found that nerve injury-induced mechanical hypersensitivity, key behavior readout of neuropathic pain, was very short-lived. Our subsequent preliminary RNA-Seq analysis identified 38 genes in the DRG, whose nerve injury-induced up regulation are significantly reduced in the CBP mutant mice. Included among these genes are Cacnalpaha2delta1and several others previously linked to nerve injury (e.g. NPY, galanin), but importantly many are not yet implicated in persistent pain and not revealed in previous microarray studies. Our proposed studies will test the hypothesis that CBP regulation of these genes is indeed critical to the persistence of neuropathic pain. In Specific Aim 1 we will validate our RNA-Seq results in a neuropathic pain model, by quantitative RT-PCR, Western blot, immunohistochemistry and in-situ hybridization. These studies will identify the subpopulations of DRG neurons (or glia/satellit cells) in which these CBP-regulated genes are expressed. In Specific Aim 2 we will assay functionality of CBP in the regulation of these genes in a neuropathic pain model, by studying promoter binding of CBP and by assaying for CBP specific histone acetylation at the promoters of Cacnalpaha2delta1 and other CBP-related genes. Finally, in Specific Aim 3 we will study neuropathic pain behavior after pharmacological inhibition of the CBP KIX domain. We will also study the effect of KIX inhibition on gene expression and epigenetic regulation of CBP-related genes in the DRG in the neuropathic pain model. Together, these studies will dissect the epigenetic landscape of chronic neuropathic pain and identify potential targets for its management. My previous research training with Drs. Eric Kandel and Howard Nash in neuroscience and molecular biology has provided me with the skills and knowledge to design, execute and analyze results of experiments. My long- term career goal, however, is to be a physician-scientist and an independent investigator studying mechanisms of chronic pain, especially the role of epigenetic regulation in the development of and recovery from chronic neuropathic pain. With this objective in mind, there are three important areas where I require additional training, mentoring and experience: (1) traditional techniques and models used in pain research, (2) cutting-edge molecular/epigenetic techniques, and (3) computational analysis of high-throughput sequencing data. In addition to gaining hands-on experience, I plan to take courses to study epigenetics and computational data analysis, at Cold Spring Harbor and at UC Berkeley. In this application I present a detailed career development plan that will enable me to acquire the additional training and mentored research experience necessary to achieve these objectives and to compete successfully for R01 funding, thereby achieving independence as a principal investigator. My department has guaranteed lab space and 80% of my professional time for my research, neither of which is contingent upon my receipt of this career award.