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.

项目总结 化学战发泡剂硫芥(SM)或其类似物氮暴露眼表(OCS) 芥末(NM)，会立即造成组织损伤和长期病理。当前的医学对策 (MCMS)常常导致疗效不完全或短暂。活性氧过度积聚 物种(ROS)诱导氧化应激(OXS)，这在OCS上皮损伤中起重要作用 各种原因，包括接触SM和类似物。ROS是由线粒体产生的。这是众所周知的 暴露于SM类似物会损害线粒体，但导致OXS的机制尚不清楚- 探索过了。最近，项目组有了一个意想不到的发现，即王朝和动态-4a，小 靶向参与线粒体动态平衡的Dynamin家族蛋白的分子具有显著的保护作用。 在OCS上皮细胞培养模型中，由于过氧化氢(HP)暴露而对抗OXS。在后续行动中 在研究中，他们发现了一种新的途径，通过抑制钙离子内流，改变细胞内钙离子的活性来保护 未折叠蛋白对动态平衡和线粒体转换孔抑制的反应 开场了。在本文提供的初步数据中，他们现在表明，NM暴露也会在 细胞培养模式，但王朝并不具有保护性。有趣的是，动力素的另一个小分子抑制剂是 保护性：Mdivi-1。值得注意的是，在NM暴露的细胞中，mdivi-1并没有使UPR趋向动态平衡 没有预防Hp感染。这些结果表明，接触后OXS的发生机制 Hp或NM必须不同，阐明Mdivi-1的作用机制可能会提供 对NM如何破坏细胞的重要洞察。Mdivi-1被广泛认为是一种特异性的抑制因子 线粒体定位的Dynamin Drp1。然而，最近有报道称，mdivi-1也直接针对 线粒体能量学。本项目的目标是研究线粒体的机制。 NM对OCS的暴露，与HP暴露的关系，以及MDVI-1对OCS的保护机制。 该团队将采用一种成熟的人类角膜上皮细胞培养模型。为了确保科学的严谨性， 原代人角膜上皮细胞将用于在体外验证关键结果，小鼠模型将 提供体内验证。为增加创新维度，活细胞评估方法和发现 将应用的技术包括钙离子成像、线粒体成像、基于细胞的报告构建、 转基因报告鼠，RNA-seq和单细胞RNA-seq(scRNA-seq)。计划中的研究结果将 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的产生，同时对健康细胞中的ROS影响有限，使其具有独特的吸引力 作为潜在的MCM。