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 铁铁作用是一种非凋亡调节细胞死亡的铁依赖性形式，已被认为是 神经退行性疾病中的神经元死亡，例如阿尔茨海默氏病。铁毒性死亡是由于 铁介导的磷脂氢过氧化物的积累。铁和脂质的活性氧 在阿尔茨海默氏病患者的大脑中，物种都增加了，并且在 小鼠的前脑神经元通过敲除抗肿瘤性过氧化物酶的GPX4导致阿尔茨海默氏症样 症状。抗氧化剂（例如维生素E和铁螯合剂）在阿尔茨海默氏症中适度有效 疾病患者，我们将其有限的效率归因于低效力和生物分布不当。我们的目标是 确切地确定脂质过氧化物需要在何处形成促进脂肪吞作用的细胞死亡特征，并 最终，这个过程是否有助于阿尔茨海默氏病。在这项研究中，一组机械 使用刺激的拉曼光谱（SRS）成像，将在细胞中定位于细胞。 SRS成像允许对没有庞大荧光标签的活细胞中的化合物分布敏感。 相反，化合物用小的脂肪族问题标记，其中包含拉曼活性官能团，例如 迪恩斯。这些铁铁毒性抑制剂的分布将阐明需要的候选亚细胞位点 保护以抑制铁铁作用。通过比较这些化合物的分布，我们可以假设哪个 细胞器/膜是脂肪杀死亡中脂质过氧化的关键部位。然后我们将调查这些 细胞器/膜特异性调节其对铁毒性脂质过氧化的敏感性，以便为了 确定它们对铁铁作用的贡献。这将通过针对既定的亲和反 - 铁氧化蛋白到这些亚细胞位点，并评估对螺旋病诱导剂的敏感性的变化。 通过这种系统的方法，我们将确定一个或多个亚细胞组件是必不可少的 为了杀虫病。总而言之，该项目的目的是（i）确定的亚细胞定位 抑制抑制神经退行性氧化物细胞死亡的药物靶标的抑制剂，（ii）至 确定通过靶向的神经元抑制和诱导铁肽死亡的基本机制 调节对铁肽脂质过氧化的敏感性。该项目的结果将加深我们 了解这种病理性细胞死亡途径，并增强了铁铁作用的未来发展 - 抑制可能成为阿尔茨海默氏症和其他人的疾病改良治疗的化合物 神经退行性疾病。通过这个项目，学员将推进他的生物学和化学 具有新颖技术的实验室技能，发展强大的研究敏锐度，并获得科学写作和 演讲经验，都在哥伦比亚的协作和跨学科研究环境中 大学。