Abnormal gene splicing in neuropathic pain - Supplement

项目总结摘要 Cacna1b基因的选择性剪接产生了许多功能不同的电压门控Cav2.2 钙通道亚型。Cav2.2控制大鼠背角伤害性感受器终末的神经递质释放 脊髓，是用于治疗神经性疼痛的镇痛剂的关键治疗靶点。Cav2.2抑制剂是 但其治疗效果复杂，缺乏广泛的疗效，治疗范围狭窄 视窗，偏离目标动作和上瘾。在伤害性感受器中，Cav2.2前-mRNA的细胞特异性选择性剪接 产生对吗啡具有不同敏感性的异构体。Cav2.2的正常剪接在 周围神经损伤后的伤害性感受器，导致有充分证据的吗啡疗效丧失 神经性疼痛。在初步实验中，申请人证明了基因组DNA的表观遗传修饰 在伤害性感受器中控制Cacna1b基因中选择性剪接外显子的细胞特异性表达。这 神经损伤后基因组DNA的修饰发生改变，导致异常选择性剪接。申请人 提出这是神经病理性疼痛的病理生理学基础上的关键改变。在这份提案中，他 计划扩大他的研究，以确定全基因组伤害性感受器特定的选择性剪接事件 在神经病理性疼痛的动物模型中被破坏。申请者将产生高分辨率、全基因组 用于识别伤害感受器特定剪接异构体的RNA-seq数据集。此外，他将确定表观遗传学 与选择性剪接相关并控制选择性剪接的DNA修饰。他将进一步展示这些 应用全基因组亚硫酸氢盐测序技术和 芯片顺序他将使用这些数据集来确定导致伤害性感受器异常选择性剪接的事件 因此，为纠正替代剪接治疗神经病理性疼痛的缺陷提供了策略。 申请者的长期职业目标是成为学术界的一名独立科学家，专注于 转录组-表观遗传相互作用如何驱动神经元中细胞特异性基因的表达 在慢性疼痛等疾病中受到干扰。为了实现这些目标，他将进行广泛的培训 大数据集的生物信息学和计算分析。这次新的培训将补充他的背景。 在膜片钳电生理学、细胞和分子生物学和行为分析方面。布朗大学 环境与建议的导师和顾问相结合为他的科学成长提供了最佳途径 和职业发展。这项培训补助金将允许申请者在不同的研究领域之间架起桥梁 了解特定于细胞的处理过程，并成为一名有竞争力的跨学科调查者。

PROJECT SUMMARY ABSTRACT Alternative splicing of the Cacna1b gene generates a number of functionally different voltage gated CaV2.2 calcium channel isoforms. CaV2.2 controls neurotransmitter release at nociceptor terminals in the dorsal horn of the spinal cord and is a key therapeutic target of analgesics used to treat neuropathic pain. CaV2.2 inhibitors are analgesic but their therapeutic effectiveness are complicated by the lack of broad efficacy, narrow therapeutic window, off target actions and addiction. In nociceptors, cell-specific alternative splicing of CaV2.2 pre-mRNA generates isoforms that have different sensitivities to morphine. Normal splicing of CaV2.2 is disrupted in nociceptors following peripheral nerve injury, contributing to the well documented loss of morphine efficacy in neuropathic pain. In preliminary experiments, the applicant shows that epigenetic modification of genomic DNA controls the cell-specific expression of an alternatively spliced exon in the Cacna1b gene in nociceptors. This modification of genomic DNA is altered after nerve injury leading to abnormal alternative splicing. The applicant proposes that this is a key alteration underlying the pathophysiology of neuropathic pain. In this proposal, he plans to expand on his studies to identify genome-wide nociceptor-specific alternative splicing events that are disrupted in an animal model of neuropathic pain. The applicant will generate high-resolution, genome-wide RNA-seq datasets to identify nociceptor-specific splice isoforms. Additionally, he will determine epigenetic modifications of DNA that associated with and control alternative splicing. He will further demonstrate how these are altered in nociceptors after nerve injury applying the techniques of whole genome bisulfite sequencing and ChIP-seq. He will use these datasets to determine events that lead to aberrant alternative splicing in nociceptors and, as a consequence, inform strategies to correct deficits in alternative splicing to treat neuropathic pain. The applicant long-term career goal is to become an independent scientist in academia focusing his research in how transcriptome-epigenetic interactions drives cell-specific gene expression in neurons and how they are disrupted in diseases such chronic pain. To achieve these goals, he will undertake extensive training in bioinformatic and computational analysis of large data sets. This new training will complement his background in patch-clamp electrophysiology, cellular and molecular biology and behavioral analyses. The Brown University environment combined with proposed mentors and consultants provides the best path for his scientific growth and career development. This training grant will allow the applicant to bridge different research areas to understand cell-specific processing, and be a competitive and interdisciplinary investigator.