Abnormal gene splicing in neuropathic pain

Cacna 1b基因的选择性剪接产生许多功能不同的电压门控CaV2.2钙通道亚型。CaV2.2控制脊髓背角中伤害感受器末端的神经递质释放，并且是用于治疗神经性疼痛的镇痛剂的关键治疗靶标。CaV2.2抑制剂是镇痛剂，但它们的治疗效果由于缺乏广泛的功效、狭窄的治疗窗、脱靶作用和成瘾而变得复杂。在伤害感受器中，CaV2.2前体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同种型。周围神经损伤后，伤害感受器中CaV2.2的正常剪接被破坏，导致吗啡在神经性疼痛中的疗效丧失。在初步实验中，申请人表明基因组DNA的表观遗传修饰控制伤害感受器中Cacna 1b基因中可变剪接外显子的细胞特异性表达。这种基因组DNA的修饰在神经损伤后改变，导致异常的选择性剪接。申请人提出这是神经性疼痛病理生理学基础的关键改变。在这项提议中，他计划扩大他的研究，以确定在神经性疼痛的动物模型中被破坏的全基因组伤害感受器特异性选择性剪接事件。申请人将生成高分辨率的全基因组RNA-seq数据集，以鉴定伤害感受器特异性剪接异构体。此外，他将确定与选择性剪接相关并控制选择性剪接的DNA表观遗传修饰。他将进一步证明这些是如何改变伤害感受器神经损伤后应用全基因组亚硫酸氢盐测序和ChIP-seq技术。他将使用这些数据集来确定导致伤害感受器中异常选择性剪接的事件，并因此为纠正选择性剪接缺陷以治疗神经性疼痛的策略提供信息。申请人的长期职业目标是成为学术界的独立科学家，重点研究转录组-表观遗传相互作用如何驱动神经元中的细胞特异性基因表达，以及它们如何在慢性疼痛等疾病中被破坏。为了实现这些目标，他将在生物信息学和大型数据集的计算分析方面进行广泛的培训。这种新的培训将补充他在膜片钳电生理学，细胞和分子生物学和行为分析的背景。布朗大学的环境与建议的导师和顾问相结合，为他的科学成长和职业发展提供了最佳途径。该培训补助金将使申请人能够弥合不同的研究领域，以了解细胞特异性处理，并成为具有竞争力的跨学科研究人员。

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 is 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.