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.

Ferroptosis, an iron-dependent form of regulated cell death by lipid peroxidation, is postulated to be the mechanism of neurodegeneration in Alzheimer’s disease. To define the precise subcellular sites of lipid peroxide formation in ferroptosis, the distribution of anti-ferroptotic compounds will be determined by stimulated Raman spectroscopy imaging, and subsequently identified subcellular targets will be modulated to increase or decrease their sensitivity to lipid peroxidation in order to characterize their role in ferroptotic death. The results of this project will provide a deeper understanding of the ferroptotic death process, and facilitate future development of targeted anti-ferroptotic compounds as potential Alzheimer’s therapies.