Mitochondria and Mustard Damage at the Ocular Surface - PROJECT SUMMARY Ocular surface (OcS) exposure to the chemical warfare vesicant sulfur mustard (SM), or its analogue nitrogen mustard (NM), causes immediate tissue damage and long-term pathology. Current medical countermeasures (MCMs) frequently result in incomplete or transient efficacy. Excessive accumulation of reactive oxygen species (ROS) induces oxidative stress (OXS), which plays prominently in OcS epithelial damage from a variety of causes, including exposure to SM and analogues. ROS are produced by mitochondria. It is known that mitochondria are damaged by exposure to SM analogues, but mechanisms leading to OXS are under- explored. Recently, the project team made the unexpected discovery that dynasore and dyngo-4a, small molecules that target dynamin family proteins involved in mitochondrial homeostasis, are remarkably protective against OXS due to hydrogen peroxide (HP) exposure in an OcS epithelial cell culture model. In a follow-up study, they identified a novel pathway whereby dynasore protects by inhibiting Ca2+ influx, shifting activity of the unfolded protein response (UPR) towards homeostasis and inhibiting mitochondrial transition pore (mPTP) opening. In Preliminary Data presented herein, they now show that NM exposure also induces the UPR in the cell culture model, but dynasore is not protective. Interestingly, another small molecule inhibitor of dynamins is protective: mdivi-1. Signficantly, mdivi-1 did not shift the UPR towards homeostasis in NM-exposed cells and did not protect against HP exposure. These results indicate that mechanisms leading to OXS after exposure to HP or NM must be different, and that elucidating the mechanism of mDivi-1 counteraction may provide important insight into how NM damages cells. Mdivi-1 has been widely considered to be a specific inhibitor of the mitochondrial-localized dynamin DRP1. However, it was recently reported that mdivi-1 also directly targets mitochondrial energetics. The objective of this project is to investigate mitochondrial mechanisms in OcS exposure to NM, the relationship to HP exposure, and mechanisms of OcS protection by mdivi-1. The team will employ a well-established human corneal epithelial cell culture model. To ensure scientific rigor, primary human corneal epithelial cells will be used to validate key results in vitro, and a mouse model will provide in vivo validation. To add an innovative dimension, live cell evaluative methods and discovery techniques will be applied, including Ca2+ imaging, mitochondrial imaging, cell-based reporter constructs, transgenic reporter mice, RNA-seq and single cell RNA-seq (scRNA-seq). Results of the planned study will provide new knowledge about how NM exposure leads to OXS. Mdivi-1 has the unusual capacity to attenuate pathological ROS production while having limited impact on ROS in healthy cells, making it uniquely attractive as a potential MCM.
