Abnormal gene splicing in neuropathic pain - Supplement

神经性疼痛中的异常基因剪接 - 补充

• 批准号: 10404737
• 负责人: Eduardo Javier Lopez Soto
• 金额: $ 9.15万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2022-01-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10404737
• 关键词: Academia; Acute; Alternative Splicing; Analgesics; Animal Model; Area; Behavioral; Bioinformatics; Calcium Channel; Cells; ChIP-seq; Complement; Computer Analysis; DNA Modification Process; Data Set; Development; Disease; Electrophysiology (science); Environment; Epigenetic Process; Event; Exons; Functional disorder; Gene Abnormality; Gene Expression; Genes; Genomic DNA; Goals; Grant; Growth and Development function; International; Lead; Mentors; Modification; Molecular; Molecular and Cellular Biology; Morphine; Neurons; Nociceptors; Pain; Pathologic; Peripheral nerve injury; Protein Isoforms; RNA Splicing; Research; Research Personnel; Resolution; Scientist; Spinal Ganglia; Spinal cord posterior horn; Spliced Genes; Techniques; Therapeutic; Training; Transcript; Universities; addiction; bisulfite sequencing; career; career development; chronic pain; experimental study; genome-wide; inhibitor/antagonist; large datasets; mRNA Precursor; nerve damage; nerve injury; neurotransmitter release; novel therapeutics; painful neuropathy; patch clamp; public health priorities; therapeutic effectiveness; therapeutic target; transcriptome; transcriptome sequencing; voltage; whole genome

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.

项目摘要摘要 CACNA1b基因的替代剪接产生了许多功能上不同的电压门cav2.2 钙通道同工型。 CAV2.2控制神经递质在伤害感受器终端的释放 脊髓是用于治疗神经性疼痛的镇痛药的关键治疗靶标。 CAV2.2抑制剂 止痛药，但由于缺乏广泛的疗效，狭窄的治疗性，其治疗效果使其具有复杂性 窗口，关闭目标动作和成瘾。在伤害感受器中，Cav2.2前MRNA的细胞特异性替代剪接 生成对吗啡具有不同敏感性的同工型。 Cav2.2的正常剪接在中断 外周神经损伤后的伤害感受器，导致有充分的吗啡疗效的丧失 神经性疼痛。在初步实验中，申请人表明基因组DNA的表观遗传学修饰 控制伤害感受器中CACNA1b基因中剪接外显子的细胞特异性表达。这 神经损伤后，基因组DNA的修饰会改变导致异常替代剪接。申请人 提出这是神经性疼痛的病理生理学基础的关键改变。在这个建议中，他 计划扩大他的研究，以确定全基因组伤害感受器特定的替代剪接事件 在神经性疼痛的动物模型中破坏。申请人将产生高分辨率，全基因组 RNA-Seq数据集识别伤害感受器特异性的剪接同工型。此外，他将确定表观遗传 与与替代剪接相关的DNA的修改。他将进一步证明这些 神经损伤后的伤害感受器改变了全基因组硫酸盐测序的技术和 chip-seq。他将使用这些数据集确定导致伤害感受器异常替代剪接的事件 因此，为纠正替代剪接的缺陷而告知策略，以治疗神经性疼痛。 申请人的长期职业目标是成为学术界的独立科学家，重点是 转录组 - 观察表相互作用如何驱动神经元中细胞特异性基因的表达及其如何 患有这种慢性疼痛的疾病破坏。为了实现这些目标，他将接受广泛的培训 大数据集的生物信息学和计算分析。这种新培训将补充他的背景 在斑块钳电生理学，细胞和分子生物学以及行为分析中。布朗大学 环境与拟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。该培训赠款将使申请人能够桥接不同的研究领域 了解特定细胞的处理，并成为竞争性和跨学科研究者。