Abnormal Gene Splicing in Neuropathic Pain

神经性疼痛中的异常基因剪接

基本信息

批准号：
10650848
负责人：
Eduardo Javier Lopez Soto
金额：
$ 24.72万
依托单位：
NORTH CAROLINA STATE UNIVERSITY RALEIGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10650848
关键词：
Academia Acute Alternative Splicing Analgesics Animal Model Area Award Behavioral Binding Bioinformatics CRISPR/Cas technology Calcium Calcium Channel Cells Cellular biology ChIP-seq Complement Computational Biology Computer Analysis DNA Binding DNA Methylation DNA Modification Process DNA-Binding Proteins Data Data Set Development Disease Effectiveness Electrophysiology (science)Environment Epigenetic Process Event Exhibits Exons Family Fellowship Functional disorder Funding Gene Abnormality Gene Expression Gene Family Genes Genomic DNA Goals Grant Growth and Development function In Vitro International Kinetics Maps Mentors Methods Modification Molecular Molecular Biology Morphine Mus Neurons Nociceptors Opioid Receptor Pain Pathologic Pathology Pattern Peripheral nerve injury Phase Phenotype Property Protein Isoforms RNA Polymerase II RNA Splicing RNA-Binding Proteins Regulation Research Research Personnel Resolution Scientist Spinal Ganglia Spinal cord posterior horn Spliced Genes Techniques Therapeutic Training Transcript Universities Viral addiction bisulfite sequencing career career development chronic pain epigenetic marker experimental study genome-wide genome-wide analysis human disease improved in vivo inhibitor injured large datasets mRNA Precursor mouse model nerve damage nerve injury nervous system disorder neurotransmitter release novel therapeutics painful neuropathy patch clamp public health priorities recruit skills spared nerve success therapeutic effectiveness therapeutic target tool transcriptome transcriptome sequencing voltage whole genome

项目摘要

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.

CACNA1b基因的替代剪接会产生许多功能上不同的电压门控CAV2.2钙通道同工型。 CAV2.2控制脊髓背角的伤害感受器末端的神经递质释放，是用于治疗神经性疼痛的镇痛药的关键治疗靶标。 CAV2.2抑制剂具有止痛药，但由于缺乏广泛的疗效，狭窄的治疗窗口，降低目标作用和成瘾，它们的治疗效果使其治疗效率变得复杂。在伤害感受器中，Cav2.2前MRNA的细胞特异性替代剪接产生对吗啡敏感性不同的同工型。周围神经损伤后，伤害感受器中CAV2.2的正常剪接受到破坏，导致神经性疼痛中有充分的吗啡疗效丧失。在初步实验中，申请人表明，基因组DNA的表观遗传修饰控制了伤害感受器中CACNA1B基因中剪接外显子的细胞特异性表达。神经损伤后，基因组DNA的这种修饰会改变导致异常替代剪接。申请人建议这是神经性疼痛病理生理学的基础的关键改变。在此提案中，他计划扩大研究，以识别全基因组伤害性特异性剪接事件，这些剪接事件在神经性疼痛的动物模型中受到破坏。申请人将生成高分辨率，全基因组RNA-seq数据集，以识别伤害感受器特异性的剪接同工型。此外，他将确定与和控制替代剪接相关的DNA的表观遗传修饰。他将进一步证明在神经损伤后使用整个基因组亚硫酸盐测序和CHIP-SEQ的技术后，伤害感受器如何改变它们。他将使用这些数据集来确定导致伤害感受器异常替代剪接的事件，并因此告知策略以纠正替代剪接中的缺陷以治疗神经性疼痛。申请人的长期职业目标是成为学术界的独立科学家，将他的研究重点放在转录组 - 概况相互作用如何驱动神经元中细胞特异性基因的表达以及如何在这种慢性疼痛的疾病中破坏它们。为了实现这些目标，他将在大型数据集的生物信息学和计算分析中接受广泛的培训。这种新的训练将补充他在斑块钳电生理学，细胞和分子生物学以及行为分析中的背景。布朗大学的环境以及拟议的导师和顾问为他的科学成长和职业发展提供了最佳途径。该培训赠款将使申请人能够桥接不同的研究领域，以了解细胞特定的处理，并成为竞争性和跨学科研究者。