Corneal and Schwann cell mechanisms in vesicant injury

Abstract Chemical threat agents can potentially be weaponized to cause mass casualties. The warfare agent sulfur mustard and its chemical analog nitrogen mustard (NM) cause severe injury to ocular tissues with acute epithelial defects, pain and delayed onset of keratitis. As yet, a comprehensive understanding of the cellular contributors and pathophysiological molecular mechanisms of corneal pathology and sensory dysfunction is lacking. Recently, we have described remarkable changes in citrullination, a protein posttranslational modification (PTM), manifests in NM-injured murine corneas. Citrullination is catalyzed by peptidyl arginine deiminases (PADs). Our preliminary findings show that PAD4 could be driving citrullination in the corneal epithelium and stroma, but its citrullinated targets have yet to be formally identified. In addition, it is still unclear how vesicant injury affects corneal sensory function and what molecular pathways in corneal Schwann cells (cSCs) contribute to axonal degeneration and regeneration. To study such complex facets of corneal pathology- structural integrity and sensory function – in this R01 grant proposal, we will investigate the epithelium and sensory system, using citrullinomics and transcriptomics approaches, respectively. Specifically in Aim 1, we will investigate what proteins are temporally altered by PAD4- driven citrullination to illuminate biological pathways and cellular processes affected by citrullinated proteins. These protein hits will be validated and assayed for function in human corneal epithelial cell culture experiments. In Aim 2, we will focus on a newly identified druggable target in cSCs whose pharmacological inhibition in NM injury promotes cSCs and axonal network regeneration and sensory recovery, and hence understanding this SC-target’s mechanisms in cSCs would be important. We will exploit a single nuclear RNA sequence analysis paradigm to pursue temporal transcriptomic changes occurring in injured corneas to illuminate novel effectors of this regenerative paradigm. Bioinformatic approaches will be used to interrogate these data to achieve a comprehensive overview of the pathways affected by citrullination mediated by PAD4 in the cornea, and a similar decoding of transcriptomic information for understanding cSC regenerative function. Ultimately, this two-pronged approach could illuminate new biomarkers and effectors of corneal injury and unravel repair mechanisms that have not been previously established.