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

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2024 · $568,821

## Abstract

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 ef...

## Key facts

- **NIH application ID:** 10933534
- **Project number:** 5R01NS076511-12
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Hülya Bayir
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $568,821
- **Award type:** 5
- **Project period:** 2012-07-01 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10933534

## Citation

> US National Institutes of Health, RePORTER application 10933534, Lipid Imaging in Traumatic Brain Injury by High Resolution GCIB-secondary Ion Mass Spectrometry (5R01NS076511-12). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10933534. Licensed CC0.

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