Epigenetic regulation in neuropathic pain

神经病理性疼痛的表观遗传调控

• 批准号: 8722049
• 负责人: Zhonghui Guan
• 金额: $ 18.94万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722049
• 关键词: Acute; Address; Affect; American; Analgesics; Antibodies; Area; Attenuated; Award; Behavior; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Calcium Channel; Cell membrane; Cells; Computer Analysis; Cyclic AMP-Responsive DNA-Binding Protein; Data; Data Analyses; Development; Development Plans; Epigenetic Process; Family; Funding; Galanin; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Goals; High-Throughput Nucleotide Sequencing; Histone Acetylation; Histones; Hyperalgesia; Hypersensitivity; Immunohistochemistry; In Situ Hybridization; Knowledge; Life; Link; Measures; Mechanics; Mentors; Mind; Modeling; Molecular; Molecular Biology; Mus; Mutant Strains Mice; Mutate; Nervous system structure; Neuroglia; Neurons; Neurosciences; Pain; Pain Research; Peripheral nerve injury; Persistent pain; Physicians; Physiological; Principal Investigator; Process; Protein Binding; Recovery; Regulation; Research; Research Personnel; Research Training; Reverse Transcriptase Polymerase Chain Reaction; Role; Scientist; Spinal Ganglia; Stimulus; Study models; Techniques; Testing; Therapeutic Intervention; Time; Training; Up-Regulation; Western Blotting; activating transcription factor; allodynia; behavior test; career; career development; chromatin immunoprecipitation; chronic neuropathic pain; chronic pain; crosslink; design; experience; gabapentin; in vivo; inhibitor/antagonist; member; nerve injury; novel; pain behavior; painful neuropathy; prevent; promoter; research study; response; satellite cell; skills; transcription factor; transcriptome sequencing

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.

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适应不良可塑性，这在生理、结构、生化和分子水平上表现出来。其结果是，对通常无害的刺激产生持续的疼痛（异常性疼痛），对通常疼痛的刺激产生过度的疼痛（痛觉过敏）。许多这些变化是基因表达持久改变的产物和/或原因。例如，之前的微阵列研究和我们的初步RNA-seq分析发现，在背根神经节（DRG）中，神经损伤会诱导钙通道alpaha2delta1亚基（Cacnalpaha2delta1）的上调，这是最常用的神经病理性止痛药加巴喷丁的目标，和 ATF3 转录因子。有趣的是，我们对 Cacnalpaha2delta1 启动子的初步分析表明它含有 ATF 结合位点。由于 ATF 家族的成员与 CBP 的 KIX 结构域结合，后者乙酰化基因启动子处的组蛋白并调节基因转录，我们测试了以下假设：CBP 通过表观遗传调节 DRG 中的基因表达，导致周围神经损伤的神经病理性疼痛后果。事实上，在 CBP KIX 结构域发生突变、导致 CBP 和转录因子（包括 ATF）之间的结合大大减少的小鼠中，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读数）非常短。住过。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突变小鼠中显着减少。这些基因包括 Cacnalpaha2delta1 和其他几个先前与神经损伤有关的基因（例如 NPY、甘丙肽），但重要的是，许多基因尚未与持续性疼痛有关，并且在之前的微阵列研究中也没有揭示。我们提出的研究将检验以下假设：CBP 对这些基因的调节确实对神经性疼痛的持续存在至关重要。在具体目标 1 中，我们将通过定量 RT-PCR、蛋白质印迹、免疫组织化学和原位杂交在神经病理性疼痛模型中验证我们的 RNA-Seq 结果。这些研究将鉴定表达这些 CBP 调节基因的 DRG 神经元（或神经胶质细胞/卫星细胞）亚群。在具体目标 2 中，我们将通过研究 CBP 的启动子结合以及测定 Cacnalpaha2delta1 和其他 CBP 相关基因启动子处的 CBP 特异性组蛋白乙酰化来测定 CBP 在神经性疼痛模型中调节这些基因的功能。最后，在具体目标 3 中，我们将研究 CBP KIX 结构域药物抑制后的神经性疼痛行为。我们还将研究 KIX 抑制对神经病理性疼痛模型中 DRG 中 CBP 相关基因的基因表达和表观遗传调控的影响。这些研究将共同​​剖析慢性神经性疼痛的表观遗传景观，并确定其治疗的潜在目标。我之前与博士一起进行的研究培训。神经科学和分子生物学领域的埃里克·坎德尔和霍华德·纳什为我提供了设计、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医师科学家和独立研究者，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展和恢复中的作用。考虑到这一目标，我在三个重要领域需要额外的培训、指导和经验：(1) 疼痛研究中使用的传统技术和模型，(2) 尖端分子/表观遗传学技术，以及 (3) 计算分析高通量测序数据。除了获得实践经验外，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，该计划将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 资助，从而实现作为首席研究员的独立性。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，这两者都不取决于我是否获得此职业奖。