Epigenetic regulation in neuropathic pain

描述(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TE神经系统的非适应性可塑性，这在生理、结构、生化和分子水平上都很明显。其结果是，对正常无害的刺激有持续性的疼痛(痛觉异常)，对正常的疼痛刺激有夸大的疼痛(痛觉过敏)。这些变化中的许多都是基因表达持久变化的产物和/或原因。例如，以前的微阵列研究和我们的初步RNA-seq分析发现，在背根节(DRG)，神经损伤诱导钙通道alpaha2delta1亚单位(Cacnalpaha2delta1)和ATF3转录因子上调，该亚单位是最常用的神经病理性止痛药加巴喷丁的靶标。有趣的是，我们对Cacnalpaha2delta1启动子的初步分析表明，它包含一个ATF结合位点。由于ATF家族的成员结合CBP的KIX结构域，在基因启动子上乙酰化组蛋白并调节基因转录，我们检验了CBP通过表观遗传调控DRG中的基因表达而参与周围神经损伤的神经病理性疼痛后果的假说。事实上，在CBP KIX结构域发生突变，导致CBP与包括ATF在内的转录因子之间的结合大大降低的小鼠中，我们发现神经损伤诱导的机械过敏是非常短暂的，这是神经病理性疼痛的关键行为读数。我们随后的初步RNA-Seq分析确定了DRG中的38个基因，其神经损伤诱导的上调在CBP突变小鼠中显著减少。这些基因包括Cacnalpaha2delta1和其他几个以前与神经损伤有关的基因(如NPY、Galanin)，但重要的是，许多基因尚未与持续性疼痛有关，也没有在以前的微阵列研究中发现。我们建议的研究将检验CBP对这些基因的调节确实对神经病理性疼痛的持久性至关重要的假设。在具体目标1中，我们将通过定量RT-PCR、Western印迹、免疫组织化学和原位杂交来验证我们在神经病理性疼痛模型中的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)的亚群。在具体目标2中，我们将通过研究CBP的启动子结合以及Cacnalpaha2delta1和其他CBP相关基因启动子上CBP特异性的组蛋白乙酰化来测试CBP在神经病理性疼痛模型中调控这些基因的功能。最后，在具体目标3中，我们将研究药物抑制CBP KIX结构域后的神经病理性疼痛行为。我们还将研究抑制KIX对神经病理性疼痛模型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病理性疼痛的表观遗传学图景，并确定其管理的潜在靶点。我之前与Eric Kandel博士和Howard Nash博士在神经科学和分子生物学方面的研究培训为我提供了设计、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员，研究慢性疼痛的机制，特别是表观遗传调节在慢性神经性疼痛的发展和康复中的作用。考虑到这一目标，我需要在三个重要领域进行额外的培训、指导和经验：(1)用于疼痛研究的传统技术和模型，(2)尖端分子/表观遗传学技术，以及(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，使我能够获得实现这些目标所需的额外培训和指导研究经验，并成功竞争R01资金，从而实现作为主要研究人员的独立性。我的部门保证了实验室空间和80%的专业时间用于我的研究，这两者都不是我获得这个职业奖项的条件。

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.