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 铁下垂是一种铁依赖的非凋亡调节的细胞死亡形式，已被认为是导致 阿尔茨海默病等神经退行性疾病中的神经元死亡。铁眼症的死亡是由于一种 铁诱导的磷脂过氧化氢不受控制的堆积。铁和脂基活性氧 阿尔茨海默病患者大脑中的物种都增加了，而在阿尔茨海默病患者中诱导的铁性下垂 小鼠的前脑神经元通过敲除Gpx4，一种抗铁上链过氧化物酶，导致阿尔茨海默病样 症状。维生素E和铁络合剂等抗氧化剂对阿尔茨海默氏症有适度的疗效 我们将其疗效有限归因于低效力和不适当的生物分布。我们的目标是 准确地定义需要在哪里形成过氧化脂质以驱动铁性下垂的细胞死亡特征，以及 最终，这个过程是否会导致阿尔茨海默氏症。在这项研究中，一组机械地 不同的铁下垂抑制剂将使用受激拉曼光谱(SRS)成像技术在细胞中定位。 SRS成像允许在没有笨重的荧光标记的情况下敏感地检测活细胞中的化合物分布； 化合物用含有拉曼活性官能团的小分子脂肪族探针标记，如 戴因斯。这些铁下垂抑制剂的分布将照亮需要的候选亚细胞部位 保护以抑制铁性下垂。通过比较这些化合物的分布，我们可以假设 细胞器/膜是铁中毒死亡过程中脂质过氧化的关键部位。然后我们将对这些进行调查 细胞器/膜通过特定地调节它们对铁性脂质过氧化的敏感性，以便 确定它们对上睑下垂的影响。这将通过将既定的支持和反对 铁下垂蛋白对这些亚细胞位置的影响，并评估对铁下垂诱导剂敏感性的变化。 通过这种系统化的方法，我们将确定一个或多个亚细胞组件是否是必不可少的 铁眼镜蛇的死亡。综上所述，本项目的目的是(I)确定脑细胞的亚细胞定位 铁下垂抑制剂，以确定抑制神经退行性氧化细胞死亡的药物靶点，以及(Ii) 通过靶向确定抑制和诱导神经元铁中毒死亡的必要膜 对铁性脂质过氧化的敏感性的调节。该项目的成果将加深我们的 了解这一病理性的细胞死亡途径，以及加强铁下垂的未来发展- 抑制化合物可能成为阿尔茨海默氏症和其他疾病的修改治疗方法 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 具有新技术的实验室技能，发展强大的研究敏锐性，并获得科学写作和 演示体验，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。