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.
Pathological pain is a major international public health priority and there is an acute need for effective and non- addictive treatments; progress toward this goal depends critically on a comprehensive understanding of its underlying molecular mechanisms. The focus of my proposal is to identify key epigenetic mechanisms that control transcript identity in nociceptors of dorsal root ganglia; nociceptors are neurons that undergo abnormal changes in activity following nerve damage and these changes are associated with the pathophysiology of neuropathic pain. The long-term goal is to inform development of new therapies to mitigate neuropathic pain.
