Lipid Imaging in Traumatic Brain Injury by High Resolution GCIB-secondary Ion Mass Spectrometry

Traumatic brain injury (TBI) is a major cause of death and disability across all ages. Secondary injury mechanisms of phospholipid (PL) peroxidation and cell death are implied in the expansion of the damage caused by the primary insult. We discovered that a required step in necrotic cell death, ferroptosis, triggered by TBI is peroxidation of arachidonoyl-phosphatidylethanolamine (A-PE) catalyzed by enzymatic complex of 15-lipoxygenase (15LOX) with PE-binding protein (PEBP1). We also showed that acyl-CoA synthetase long-chain 4 (ACSL4), responsible for A-PE synthesis, is an essential positive ferroptosis regulator. However, two questions: 1) Which cell types undergo PE oxidation in ferroptosis, and 2) Why ferroptosis sensitivity depends on ACSL4 given high content of A-PE in the brain – remain unanswered due to lack of subcellular-resolution imaging of diversified lipids in different cell types. To address question 1, we developed innovative dual- secondary ion mass spectrometry (SIMS) imaging workflow whereby imaging with water gas cluster ion beams ((H2O)n-GCIB)-SIMS was combined with labeling of proteins by lanthanide conjugated antibodies (up to ~40). This allowed subcellular mapping on the same tissue section of individual PLs, including oxidized PLs (PLox) with ~100-1,000 times higher sensitivity and lateral resolution ~1µm and identificiation of cell types undergoing lipid peroxidation. To address question 2, we developed liquid chromatography-MS-based redox lipidomics approaches to quantitate minor lipid peroxidation substrates not only during the progression of ferroptosis but also at its initiation. We found that initiation occurs via peroxidation of a minor oxidizable di-Arachidonoyl-PE (di-A-PE) which has arachidonoyl residues in both sn-1 and sn-2 positions. Preliminary data show that 15LOX alone readily oxidizes di-A-PE, leading to ferroptosis initiation. Normally di-A-PE is utilized for synthesis of endocannabinoids, particularly arachidonoyl-amide (anandamide). This pathway utilizes sn-1 acyl of di-A-PE to enzymatically transacylate the amino group of another PE, yielding N-acyl-PE (NAPE). In the context of ferroptosis, the N-transacylation is a strong competitor of di-A-PE oxidation by 15-LOX. Thus, di-AA-PE synthesis, requiring ACSL4, might be a ferroptosis bottleneck. Our central hypotheses are: i) TBI-induced ferroptosis is a two-stage process initiated by di-A-PE peroxidation followed by the propagation stage where mono-A-PE species get oxidized at a lower rate; ii) di-A-PE formation is decisive factor in sensitivity to ferroptosis vs NAPE /anandamide formation after TBI. Our specific aims include: 1. Optimize SIMS imaging protocols for molecular characterization of PLox and their substrates in different cell types in normal and injured brains. 2. Determine the role of ACSL4 and NAPEs in molecular specificity of lipid peroxidation in different cell types of injured brain. 3. Utilize single cell SIMS imaging to evaluate the effectiveness of novel mechanism based selective ferroptosis inhibitors: 1) bis-allylic deuterated arachidonic acid, and 2) inhibitor of 15LOX/PEBP1 complex.