SUMMARY Ferroptosis is a form of programmed cell death pathogenic to several acute and chronic diseases and executed via oxygenation of polyunsaturated phosphatidylethanolamines (PE) by 15-lipoxygenases (15-LO) that normally use free polyunsaturated fatty acids as substrates. Mechanisms of the altered 15-LO substrate specificity are enigmatic. We sought a common ferroptosis regulator for 15LO. We discovered that PEBP1, a scaffold protein inhibitor of protein kinase cascades, complexes with two 15LO isoforms, 15LO1 and 15LO2, and changes their substrate competence to generate hydroperoxy-PE. Inadequate reduction of hydroperoxy-PE due to insufficiency or dysfunction of a selenoperoxidase, GPX4, leads to ferroptosis. We demonstrated the importance of PEBP1-dependent regulatory mechanisms of ferroptotic death in airway epithelial cells in asthma, kidney epithelial cells in renal failure and cortical and hippocampal neurons in brain trauma. As master regulators of ferroptotic cell death with profound implications for human disease, PEBP1/15LO complexes represent a new target for drug discovery.

OBJECTIVES: Traumatic brain injury (TBI) triggers multiple cell death pathways, possibly including ferroptosis -a recently described cell death pathway that results from accumulation of 15-lipoxygenase (15-LOX)-mediated lipid oxidation products, specifically oxidized phosphatidylethanolamine (PE) containing arachidonic (AA) or adrenic (AdA) acid. This study aimed to investigate whether ferroptosis contributed to the pathogenesis of in vitro and in vivo TBI, and whether inhibition of 15-LOX provided neuroprotection. DESIGN:Cell culture study and randomized, controlled animal study.

Lipid peroxidation can be broadly defined as the process of inserting a hydroperoxy group into a lipid. Polyunsaturated fatty acids present in the phospholipids are often the targets for peroxidation. Phospholipids are indispensable for normal structure of membranes. The other important function of phospholipids stems from their role as a source of lipid mediators – oxygenated free fatty acids that are derived from lipid peroxidation. In the CNS, excessive accumulation of either oxidized phospholipids or oxygenated free fatty acids may be associated with damage occurring during acute brain injury and subsequent inflammatory responses. There is a growing body of evidence that lipid peroxidation occurs after severe traumatic brain injury in humans and correlates with the injury severity and mortality. Identification of the products and sources of lipid peroxidation and its enzymatic or non-enzymatic nature is essential for the design of mechanism-based therapies. Recent progress in mass spectrometry-based lipidomics/oxidative lipidomics offers remarkable opportunities for quantitative characterization of lipid peroxidation products, providing guidance for targeted development of specific therapeutic modalities. In this review, we critically evaluate previous attempts to use non-specific antioxidants as neuroprotectors and emphasize new approaches based on recent breakthroughs in understanding of enzymatic mechanisms of lipid peroxidation associated with specific death pathways, particularly apoptosis. We also emphasize the role of different phospholipases (calcium-dependent and -independent) in hydrolysis of peroxidized phospholipids and generation of pro- and anti-inflammatory lipid mediators.

sn2-15-HpETE-PE generated by mammalian 15-lipoxygenase/phosphatidylethanolamine binding protein-1 (PEBP1) complex is a death signal in a recently identified type of programmed cell demise, ferroptosis. How the enzymatic complex selects sn2-ETE-PE as the substrate among 1 of ~100 total oxidizable membrane PUFA phospholipids is a central yet unresolved question. To unearth the highly selective and specific mechanisms of catalytic competence, we used a combination of redox lipidomics, mutational and computational structural analysis to show they stem from: i) reactivity towards readily accessible hexagonally organized membrane sn2-ETE-PEs, ii) relative preponderance of sn2-ETE-PE species vs. other sn2-ETE-PLs, iii) allosteric modification of the enzyme in the complex with PEBP1. This emphasizes the role of enzymatic vs. random stochastic free radical reactions in ferroptotic death signaling.

Oxidative stress is a major contributor to secondary injury signaling cascades following traumatic brain injury (TBI). The role of lipid peroxidation in the pathophysiology of a traumatic insult to neural tissue is increasingly recognized. As the methods to quantify lipid peroxidation have gradually improved, so has the understanding of mechanistic details of lipid peroxidation and related signaling events in the injury pathogenesis. While free-radical mediated, non-enzymatic lipid peroxidation has long been studied, recent advances in redox lipidomics have demonstrated the significant contribution of enzymatic lipid peroxidation to TBI pathogenesis. Complex interactions between inflammation, phospholipid peroxidation, and hydrolysis define the engagement of different cell death programs and the severity of injury and outcome. This review focuses on enzymatic phospholipid peroxidation after TBI, including the mechanism of production, signaling roles in secondary injury pathology, and temporal course of production with respect to inflammatory response. In light of the newly identified phospholipid oxidation mechanisms, we also discuss possible therapeutic targets to improve neurocognitive outcome after TBI. Finally, we discuss current limitations in identifying oxidized phospholipids and possible methodologic improvements that can offer a deeper insight into the region-specific distribution and subcellular localization of phospholipid oxidation after TBI.

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Traumatic brain injury (TBI) is a major health problem associated with significant morbidity and mortality. The pathophysiology of TBI is complex involving signaling through multiple cascades, including lipid peroxidation. Oxidized free fatty acids, a prominent product of lipid peroxidation, are potent cellular mediators involved in induction and resolution of inflammation and modulation of vasomotor tone. While previous studies have assessed lipid peroxidation after TBI, to our knowledge no studies have used a systematic approach to quantify the global oxidative changes in free fatty acids. In this study, we identified and quantified 244 free fatty acid oxidation products using a newly developed global liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. This methodology was used to follow the time course of these lipid species in the contusional cortex of our pediatric rat model of TBI. We show that oxidation peaked at 1 hr after controlled cortical impact and was progressively attenuated at 4 and 24 hr time points. While enzymatic and non-enzymatic pathways were activated at 1 hr post-TBI, enzymatic lipid peroxidation was the predominant mechanism with 15-lipoxygenase (LOX) contributing to the majority of total oxidized fatty acid content. Pro-inflammatory lipid mediators were significantly increased at 1 and 4 hr after TBI with return to basal levels by 24 hr. Anti-inflammatory lipid mediators remained significantly increased across all three time points, indicating an elevated and sustained anti-inflammatory response following TBI.

Objective: Brain mitochondrial dysfunction limits neurological recovery after cardiac arrest (CA). Brain polyunsaturated cardiolipins (CLs), mitochondria-unique and functionally essential phospholipids, have unprecedented diversification. Since brain CLs are not present in plasma normally, we hypothesized their appearance would correlate with brain injury severity early after CA and return of spontaneous circulation (ROSC). Design: Observational case-control study Setting: Two medical centers within one city Participants (subjects):We enrolled 41 adult cardiac arrest patients in whom blood could be obtained within 6h of resuscitation. Two subjects were excluded following outlier analysis. Ten healthy subjects were controls. Sprague-Dawley rats were used in asphyxial CA studies.