Mitochondria and Mustard Damage at the Ocular Surface

眼表的线粒体和芥末损伤

• 批准号: 10708498
• 负责人: M. Elizabeth Fini
• 金额: $ 53.74万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10708498
• 关键词: Abbreviations; Alkylation; Attenuated; Bioenergetics; Cell Culture Techniques; Cells; Chemical Warfare; Chloroquine; Chronic; Complex; Consumption; Cornea; Cysteine; Cytoprotection; Data; Development; Dimensions; Doxycycline; Dynamin; Electron Transport; Ensure; Epithelial Cells; Epithelium; Exposure to; Eye; Follow-Up Studies; Gene Expression Profile; Genes; Homeostasis; Human; Hydrogen Peroxide; Image; In Vitro; Knowledge; Mechlorethamine; Medical; Methods; Mitochondria; Modeling; Molecular; Mus; Mustard; Mustard Compounds; Mustard Gas; Names; Nomenclature; Oxidative Stress; Oxidative Stress Induction; Pathologic; Pathology; Pathway interactions; Phase; Phenotype; Play; Production; Protein Family; Proteins; Public Health; Reactive Oxygen Species; Reporter; Reporter Genes; Reporting; Route; Stress; Techniques; Tissues; Toxic effect; Toxicant exposure; Transgenic Organisms; Validation; Vesicants; analog; cell injury; chemical disaster; chemical threat; corneal epithelium; eye dryness; in vivo; inhibitor; innovation; insight; limbal; medical countermeasure; mitochondrial permeability transition pore; mouse model; novel; ocular surface; prevent; response; response to injury; single-cell RNA sequencing; small molecule; small molecule inhibitor; synergism; tert-Butylhydroperoxide; therapeutic target; transcriptome sequencing

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.

项目概要 眼表 (OcS) 暴露于化学战发泡剂硫芥 (SM) 或其类似物氮 芥末（NM）会导致立即组织损伤和长期病理。目前的医疗对策 （MCM）经常导致不完全或短暂的疗效。活性氧累积过多 ROS 会诱导氧化应激 (OXS)，氧化应激在 OcS 上皮损伤中发挥着重要作用。 多种原因，包括接触 SM 和类似物。 ROS 由线粒体产生。我们都知道 线粒体因接触 SM 类似物而受损，但导致 OXS 的机制尚不明确 探索过。近日，项目组意外发现dynasore和dyngo-4a，小型 靶向参与线粒体稳态的动力家族蛋白的分子具有显着的保护作用 在 OcS 上皮细胞培养模型中，针对由于过氧化氢 (HP) 暴露而导致的 OXS。在后续行动中 研究中，他们发现了一种新的途径，Dynasore 通过抑制 Ca2+ 流入、改变 针对稳态的未折叠蛋白反应 (UPR) 和抑制线粒体转换孔 (mPTP) 开放。在本文提供的初步数据中，他们现在表明 NM 暴露也会诱导 UPR 细胞培养模型，但 dynasore 不具有保护作用。有趣的是，另一种动力蛋白小分子抑制剂是 防护：mdivi-1。值得注意的是，mdivi-1 并未将 NM 暴露细胞中的 UPR 转向稳态，并且 不能防止 HP 暴露。这些结果表明，暴露于环境后导致 OXS 的机制 HP 或 NM 必须不同，并且阐明 mDivi-1 的反作用机制可能会提供 关于 NM 如何损害细胞的重要见解。 Mdivi-1被广泛认为是一种特异性抑制剂 线粒体定位的动力蛋白 DRP1。然而，最近有报道称，mdivi-1也直接针对 线粒体能量学。该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 的 OcS 保护机制。 该团队将采用成熟的人类角膜上皮细胞培养模型。为保证科学严谨性， 原代人角膜上皮细胞将用于体外验证关键结果，小鼠模型将 提供体内验证。添加创新维度、活细胞评估方法和发现 将应用技术，包括 Ca2+ 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 具有不同寻常的衰减能力 病理性 ROS 产生，同时对健康细胞中 ROS 的影响有限，使其具有独特的吸引力 作为一个潜在的MCM。