Elucidating the Role of Lipid Metabolism and Peroxidation in Ferroptosis

Abstract

Ferroptosis is a form of cell death that is dependent on the iron-mediated peroxidation of lipids within a cell. Since its discovery in 2012, ferroptosis has been implicated in many disease states, including neurodegeneration, ischemia reperfusion injury, atherosclerosis, and cancer, making it an attractive therapeutic target. However, much is still unknown about the regulation of ferroptosis, hindering our ability to modulate this process. Since the peroxidation of lipids within a cell is a hallmark of ferroptosis, this thesis aims to explore the role that lipid metabolism and peroxidation play in mediating ferroptotic death by examining the metabolism, oxidation, location, and genetic regulation of lipids in cells undergoing ferroptosis. In this work, we first developed a strategy to trace the metabolism of a peroxidation-prone polyunsaturated fatty acid (PUFA), arachidonic acid, in-vitro using stable isotope labeling and mass spectrometry-based lipidomics. The strategy was then applied to other common PUFAs, including conjugated and nonconjugated linoleic acid. To facilitate the identification of potential lipid peroxidation products in complex lipidomics datasets, we created an experimental oxidized lipid library by performing in-vitro peroxidation of various oxidizable lipids and characterizing the resulting products using mass spectrometry. Next, we sought to examine the subcellular location and morphological changes associated with lipid peroxidation in cells using stimulated Raman scattering (SRS) and confocal microscopy. Finally, we sought to further characterize the effect of genetic ablation of the enzyme acyl-CoA synthetase long chain family member 4, or ACSL4, which has been shown to confer robust ferroptosis protection in multiple cell types. We employed various -omics methodologies, including lipidomics, transcriptomics, sterolomics, and proteomics, to improve our understanding of the mechanism behind the ferroptosis resistance afforded by ACSL4 knockout. The collective findings and implications of this work as well as potential future directions will be discussed in the final chapter.