Identification of essential sites of lipid peroxidation in ferroptosis using Raman spectroscopy imaging

使用拉曼光谱成像鉴定铁死亡中脂质过氧化的重要位点

• 批准号: 10457460
• 负责人: Alfred Nikolai von Krusenstiern
• 金额: $ 4.74万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10457460
• 关键词: Aging; Alzheimer' s Disease; Alzheimer' s disease patient; Alzheimer' s disease therapy; Antioxidants; Arachidonate 15-Lipoxygenase; Biodistribution; Biological; Brain; Cell Death; Cells; Cellular Membrane; Cessation of life; Characteristics; Clinical; Deferoxamine; Dementia; Detection; Development; Disease; Disease Progression; Distributional Activity; Docosahexaenoic Acids; Drug Targeting; Environment; Functional disorder; Future; Glutathione; Goals; Hydrogen Peroxide; Image; Interdisciplinary Study; Iron; Iron Chelating Agents; Knockout Mice; Label; Laboratory Chemicals; Link; Lipid Peroxidation; Lipid Peroxides; Lipids; Locales; Lysosomes; Mediating; Membrane; Mitochondria; Mus; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Pathogenesis; Pathologic; Pathway interactions; Patients; Peroxidases; Persons; Pharmaceutical Preparations; Pharmacology; Phenotype; Phospholipids; Play; Predisposition; Probucol; Process; Prosencephalon; Proteins; Raman Spectrum Analysis; Reactive Oxygen Species; Research; Role; Site; Specificity; Stains; Structure; Symptoms; System; Techniques; Universities; Vitamin E; Writing; base; cancer therapy; drug candidate; drug distribution; erastin; experience; experimental study; follow-up; functional group; inhibitor; interest; lipophilicity; neuron loss; novel; overexpression; peroxidation; phenylmethylpyrazolone; prevent; skills; spectroscopic imaging; subcellular targeting; vibration

Project Summary Ferroptosis, an iron-dependent form of non-apoptotic regulated cell death, has been suggested as a cause of neuronal death in neurodegenerative disorders such as Alzheimer’s disease. Ferroptotic death is due to an uncontrolled iron-mediated accumulation of phospholipid hydroperoxides. Iron and lipid-based reactive oxygen species are both increased in the brains of patients with Alzheimer’s disease, and induction of ferroptosis in the forebrain neurons of mice by knocking out GPX4, an anti-ferroptotic peroxidase, results in Alzheimer-like symptoms. Anti-oxidants such as vitamin E and iron chelators have been moderately effective in Alzheimer’s disease patients, and we attribute their limited efficacy to low potency and improper biodistribution. Our goal is to define precisely where lipid peroxides need to form to drive the cell death characteristic of ferroptosis, and ultimately whether this process contributes to Alzheimer’s disease. In this study, a group of mechanistically distinct ferroptosis inhibitors will be localized in cells using stimulated Raman spectroscopy (SRS) imaging. SRS imaging allows sensitive detection of compound distribution in live cells without bulky fluorescent tags; compounds are instead labeled with small, aliphatic probes containing Raman-active functional groups such as diynes. The distribution of these ferroptosis inhibitors will illuminate candidate subcellular sites that require protection to inhibit ferroptosis. By comparing the distributions of these compounds, we can hypothesize which organelles/membranes are key sites of lipid peroxidation in ferroptotic death. We will then investigate these organelles/membranes by specifically modulating their sensitivity to ferroptotic lipid peroxidation, in order to determine their contribution to ferroptosis. This will be accomplished by targeting established pro- and anti- ferroptotic proteins to these subcellular sites, and evaluating changes in sensitivity to ferroptosis inducers. Through this systematic approach, we will identify whether one or more subcellular components are essential to ferroptotic death. In summary, the aims of this project are to (i) to determine the subcellular localization of ferroptosis inhibitors to identify targets of drugs inhibiting neurodegenerative oxidative cell death, and (ii) to identify the essential membranes for inhibition and induction of ferroptotic death in neurons through targeted modulation of sensitivity to ferroptotic lipid peroxidation. The results of this project will deepen our understanding of this pathological cell death pathway, as well as enhance future development of ferroptosis- inhibiting compounds that could become disease-modifying treatments for Alzheimer’s and other neurodegenerative diseases. Through this project, the trainee will advance his biological and chemical laboratory skills with novel techniques, develop a strong research acumen, and gain scientific writing and presentation experience, all in a collaborative and interdisciplinary research environment at Columbia University.

项目概要 铁死亡是一种铁依赖性非凋亡调节细胞死亡形式，已被认为是 阿尔茨海默病等神经退行性疾病中的神经元死亡。铁死亡是由于 不受控制的铁介导的磷脂氢过氧化物的积累。铁和脂基活性氧 阿尔茨海默氏病患者大脑中的物种数量增加，并且在大脑中诱导铁死亡 通过敲除 GPX4（一种抗铁死亡过氧化物酶）小鼠的前脑神经元，可导致阿尔茨海默病样症状 症状。维生素 E 和铁螯合剂等抗氧化剂对阿尔茨海默病有一定效果 疾病患者，我们将其有限的疗效归因于效力低和生物分布不当。我们的目标是 精确定义脂质过氧化物需要在何处形成以驱动铁死亡的细胞死亡特征，以及 最终这个过程是否会导致阿尔茨海默病。在这项研究中，一组机械地 使用受激拉曼光谱（SRS）成像将不同的铁死亡抑制剂定位在细胞中。 SRS 成像可以灵敏地检测活细胞中的化合物分布，无需大量荧光标签； 相反，化合物用含有拉曼活性官能团的小型脂肪族探针进行标记，例如 迪因。这些铁死亡抑制剂的分布将阐明需要的候选亚细胞位点 抑制铁死亡的保护作用。通过比较这些化合物的分布，我们可以假设哪些 细胞器/膜是铁死亡中脂质过氧化的关键部位。然后我们将调查这些 通过特异性调节细胞器/膜对铁死亡脂质过氧化的敏感性，以便 确定它们对铁死亡的贡献。这将通过针对既定的支持者和反对者来实现 铁死亡蛋白到这些亚细胞位点，并评估对铁死亡诱导剂的敏感性变化。 通过这种系统方法，我们将确定一个或多个亚细胞成分是否是必需的 至铁死亡。总之，该项目的目标是（i）确定 铁死亡抑制剂以确定抑制神经退行性氧化细胞死亡的药物靶点，以及（ii） 通过靶向识别抑制和诱导神经元铁死亡的重要膜 调节对铁死亡脂质过氧化的敏感性。该项目的成果将加深我们 了解这种病理性细胞死亡途径，并促进铁死亡的未来发展 抑制化合物可能成为阿尔茨海默病和其他疾病的疾病缓解疗法 神经退行性疾病。通过这个项目，学员将提高他的生物和化学水平 通过新颖的技术培养实验室技能，培养强大的研究敏锐度，并获得科学写作和 演讲经验，全部在哥伦比亚大学的协作和跨学科研究环境中进行 大学。