Mitochondria and Mustard Damage at the Ocular Surface

项目摘要 眼表（OcS）暴露于化学战发泡剂硫芥（SM）或其类似物氮 芥子气（NM），造成即时组织损伤和长期病理。当前的医疗对策 （MCMs）通常导致不完全或短暂的疗效。活性氧过度积累 活性氧（ROS）诱导氧化应激（OXS），这在OXS上皮损伤中起着重要作用。 各种原因，包括暴露于SM和类似物。ROS由线粒体产生。已知 暴露于SM类似物会损伤线粒体，但导致OXS的机制尚不清楚。 探讨了最近，项目组意外地发现，dynasore和dyngo-4a，小 靶向参与线粒体稳态的动力蛋白家族蛋白的分子， 在OcS上皮细胞培养模型中，由于过氧化氢（HP）暴露，OXS的抗氧化性降低。新闻和跟踪 在这项研究中，他们确定了一种新的途径，dynasore通过抑制Ca 2+内流，改变 未折叠蛋白反应（UPR）对稳态和抑制线粒体过渡孔（mPTP） 开放后在本文提供的初步数据中，他们现在表明NM暴露也诱导了 细胞培养模型，但dynasore没有保护作用。有趣的是，动力蛋白的另一种小分子抑制剂是 保护：mdivi-1。明显地，在NM暴露的细胞中，mdivi-1没有使UPR向稳态转变， 不能防止HP暴露。这些结果表明，暴露后导致OXS的机制， HP或NM必须是不同的，并且阐明mDivi-1抵消作用的机制可以提供 对NM如何损害细胞的重要见解。Mdivi-1已被广泛认为是一种特异性抑制剂， 大脑中的发动蛋白DRP 1然而，最近有报道称，mdivi-1也直接针对 线粒体能量学本项目的目的是研究线粒体机制， OcS暴露于NM，与HP暴露的关系，以及mdivi-1对OcS保护的机制。 该团队将采用一种成熟的人类角膜上皮细胞培养模型。为了确保科学严谨， 原代人角膜上皮细胞将用于验证体外关键结果，小鼠模型将 提供体内验证。为了增加一个创新的维度，活细胞评估方法和发现 技术将被应用，包括Ca 2+成像，线粒体成像，基于细胞的报告构建体， 转基因报告小鼠、RNA-seq和单细胞RNA-seq（scRNA-seq）。计划研究的结果将 提供有关NM暴露如何导致OXS的新知识。Mdivi-1具有非同寻常的能力， 病理性ROS产生，同时对健康细胞中的ROS具有有限的影响，使其具有独特的吸引力， 作为潜在的MCM。

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.