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: 10368042
• 负责人: Alfred Nikolai von Krusenstiern
• 金额: $ 5.1万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10368042
• 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; 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/antagonist; 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.

项目摘要 铁凋亡是一种铁依赖性的非凋亡调节性细胞死亡形式，已被认为是导致细胞凋亡的原因。 神经退行性疾病如阿尔茨海默病中的神经元死亡。铁中毒死亡是由于 不受控制的铁介导的磷脂氢过氧化物的积累。铁和脂质基活性氧 在阿尔茨海默病患者的大脑中，两种铁的含量都增加，并且在阿尔茨海默病患者的大脑中诱导铁凋亡。 通过敲除GPX 4（一种抗铁蛋白过氧化物酶）， 症状抗氧化剂如维生素E和铁螯合剂对阿尔茨海默氏症有一定的疗效 我们将其有限的功效归因于低效力和不适当的生物分布。我们的目标是 精确定义脂质过氧化物需要在哪里形成以驱动铁凋亡的细胞死亡特征， 最终确定这一过程是否会导致阿尔茨海默病。在这项研究中，一组机械 使用受激拉曼光谱（SRS）成像将不同的铁凋亡抑制剂定位在细胞中。 SRS成像允许灵敏地检测活细胞中的化合物分布，而无需庞大的荧光标记; 化合物用含有拉曼活性官能团的小的脂肪族探针标记， diynes。这些铁凋亡抑制剂的分布将阐明候选的亚细胞位点， 保护以抑制铁凋亡。通过比较这些化合物的分布，我们可以假设 细胞器/膜是铁中毒性死亡中脂质过氧化的关键部位。我们将调查这些 细胞器/膜通过特异性调节其对铁蛋白脂质过氧化的敏感性， 确定它们对铁下垂的贡献。这将是通过针对既定的亲，反， 这些亚细胞位点的铁凋亡蛋白，并评估对铁凋亡诱导剂敏感性的变化。 通过这种系统的方法，我们将确定是否一个或多个亚细胞成分是必不可少的 铁中毒死亡总之，本项目的目的是（i）确定 铁凋亡抑制剂，以鉴定抑制神经变性氧化性细胞死亡的药物的靶标，和（ii） 识别通过靶向的神经元铁凋亡的抑制和诱导的基本膜 调节对铁中毒脂质过氧化反应的敏感性。该项目的成果将加深我们的 了解这种病理性细胞死亡途径，以及加强铁凋亡的未来发展- 抑制化合物，可能成为阿尔茨海默氏症和其他疾病的疾病改善治疗， 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 实验室技能与新技术，发展强大的研究敏锐度，并获得科学写作和 演讲经验，所有在合作和跨学科的研究环境在哥伦比亚 大学