Epigenetic regulation in neuropathic pain

神经病理性疼痛的表观遗传调控

• 批准号: 8443366
• 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/8443366
• 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; 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; RNA; 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

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. PUBLIC HEALTH RELEVANCE: Nerve injury-induced chronic neuropathic pain is a maladaptive neuronal process that affects millions of Americans. Our proposed experiments will reveal the epigenetic and genetic contributors to this debilitating condition. Our objective is to obtain a better understanding of the molecular underpinnings of chronic neuropathic pain, and to identify novel targets for effective therapeutic intervention.

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，其在生理、结构、生物化学和分子水平上表现出来。其结果是一种病症，其中存在对正常无害刺激的持续疼痛（异常性疼痛）和对正常疼痛刺激的过度疼痛（痛觉过敏）。这些变化中的许多是基因表达持续改变的产物和/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，在背根神经节（DRG）中，神经损伤诱导钙通道alpaha 2delta 1亚基（Cacnalpaha 2delta 1）的上调，这是最常用的神经性疼痛药物加巴喷丁和ATF 3转录因子的靶向。有趣的是，我们对Cacnalpaha 2delta 1启动子的初步分析表明，它含有ATF结合位点。由于ATF家族成员与CBP的KIX结构域结合，从而使基因启动子处的组蛋白乙酰化并调节基因转录，因此我们测试了CBP通过表观遗传学调节背根神经节中的基因表达而导致周围神经损伤的神经性疼痛后果的假设。事实上，在CBP KIX结构域突变的小鼠中，CBP和转录因子（包括ATF）之间的结合大大减少，我们发现神经损伤诱导的机械超敏反应（神经性疼痛的关键行为读出）非常短暂。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突变小鼠中显著降低。这些基因中包括Cacnalpaha 2delta 1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，也未在先前的微阵列研究中发现。我们提出的研究将检验这样一个假设，即CBP对这些基因的调控确实对神经性疼痛的持续性至关重要。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。这些研究将确定这些CBP调控基因表达的DRG神经元（或神经胶质/卫星细胞）的亚群。在特定目标2中，我们将通过研究CBP的启动子结合以及通过测定Cacnalpaha 2delta 1和其他CBP相关基因启动子处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，我们将研究药物抑制CBP KIX结构域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。总之，这些研究将剖析慢性神经性疼痛的表观遗传景观，并确定其管理的潜在目标。我之前在神经科学和分子生物学方面与Eric Kandel博士和霍华德纳什博士进行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医生科学家和一名独立的研究者，研究慢性疼痛的机制，特别是表观遗传调节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，我需要额外的培训，指导和经验的三个重要领域：（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。在本申请中，我提出了一个详细的职业发展计划，这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R 01资金，从而实现作为主要研究者的独立性。我的部门保证实验室空间和我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这个职业奖。 公共卫生相关性：神经损伤引起的慢性神经性疼痛是一种适应不良的神经过程，影响数百万美国人。我们提出的实验将揭示这种衰弱状况的表观遗传和遗传因素。我们的目标是更好地了解慢性神经病理性疼痛的分子基础，并确定有效的治疗干预的新靶点。