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.
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.