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

创伤性脑损伤（TBI）是所有年龄段死亡和残疾的主要原因。继发性损伤 磷脂（PL）过氧化和细胞死亡的机制被暗示在损害的扩大 造成的伤害我们发现坏死细胞死亡的一个必要步骤，铁凋亡， 由TBI引起的是花生四烯酸-磷脂酰乙醇胺（A-PE）的过氧化反应， 15-脂氧合酶（15 LOX）与PE结合蛋白（PEBP 1）。我们还发现酰基辅酶A合成酶 长链4（ACSL 4），负责A-PE合成，是一种重要的正性铁凋亡调节剂。 然而，两个问题：1）哪些细胞类型在铁凋亡中经历PE氧化，以及2）为什么铁凋亡 鉴于大脑中A-PE含量高，敏感性取决于ACSL 4-由于缺乏 亚细胞分辨率成像的多样化脂质在不同的细胞类型。为了解决问题1，我们 开发了创新的双二次离子质谱（西姆斯）成像工作流程， 采用水气团簇离子束（H_2O）n-GCIB）-西姆斯结合镧系元素标记蛋白质 偶联抗体（高达~40）。这允许在相同的组织切片上进行亚细胞定位， 单个PL，包括灵敏度和横向分辨率提高约100- 1，000倍的氧化PL（PLox） ~1µm，并鉴定发生脂质过氧化的细胞类型。为了解决问题2，我们开发了 基于液相色谱-质谱的氧化还原脂质组学方法定量测定轻微脂质过氧化 不仅在铁凋亡的进展过程中，而且在其开始时也是底物。我们发现入会仪式 通过具有花生四烯酰基残基的少量可氧化二-花生四烯酰基-PE（二-A-PE）的过氧化作用发生 在SN-1和SN-2位置上。初步数据显示，15 LOX单独容易氧化二-A-PE，导致 到铁性下垂的发生通常，二-A-PE用于合成内源性大麻素，特别是 花生四烯酸酰胺（anandamide）。该途径利用二-A-PE的sn-1酰基酶促转酰化 另一个PE的氨基，产生N-酰基-PE（NAPE）。在铁凋亡的背景下，N-转酰化 是15-LOX氧化di-A-PE的强有力竞争者。因此，需要ACSL 4的二-AA-PE合成可能是 一个铁性下垂的瓶颈我们的中心假设是：i）TBI诱导的铁凋亡是一个两阶段的过程 由二-A-PE过氧化反应引发，随后是单-A-PE物质被氧化的增殖阶段 ii）二-A-PE形成是对铁凋亡相对于NAPE /花生四烯酸的敏感性的决定性因素 TBI后形成。我们的具体目标包括：1.优化西姆斯成像方案， PLox及其底物在正常和受损脑中的不同细胞类型中的表征。2.确定 ACSL 4和NAPE在不同损伤细胞脂质过氧化反应分子特异性中作用 个脑袋3.利用单细胞西姆斯成像来评估基于新机制的选择性 铁凋亡抑制剂：1）双烯丙基氘代花生四烯酸，和2）15 LOX/PEBP 1复合物的抑制剂。

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.