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

创伤性脑损伤(TBI)是所有年龄段的死亡和残疾的主要原因。继发性损伤 磷脂过氧化和细胞死亡的机制暗示了损伤的扩大 由最初的侮辱造成的。我们发现，坏死性细胞死亡的一个必要步骤，铁性下垂，引发了 以花生四氢叶酸-磷脂酰乙醇胺(A-PE)为底物，催化A-PE过氧化。 15-脂氧合酶(15LOX)和PE结合蛋白(PEBP1)。我们还发现，酰基辅酶A合成酶 长链4(ACSL4)负责A-PE的合成，是一种重要的正性铁下垂调节剂。 然而，有两个问题：1)在铁性下垂中，哪些细胞类型经历PE氧化；2)为什么发生铁性下垂 由于大脑中A-PE含量较高，敏感性取决于ACSL4-由于缺乏A-PE而仍未得到回答 不同细胞类型的各种脂类的亚细胞分辨率成像。为了回答问题1，我们 开发创新的双二次离子质谱仪(SIMS)成像工作流程 用水煤气团簇离子束((H2O)n-GCIB)-SIMS结合稀土元素标记蛋白质 结合抗体(最多40个)。这使得在同一组织切片上进行亚细胞定位成为可能。 单独的PL，包括氧化的PL(PLOX)，灵敏度和横向分辨率提高~100-1000倍 ~1微米，并鉴定正在经历脂质过氧化的细胞类型。为了解决第二个问题，我们开发了 基于液质联用的氧化还原脂质组学方法定量检测轻微的脂质过氧化 底物不仅在铁性下垂的进展过程中，而且在其开始的时候。我们发现那次入会 通过少量可氧化的具有花生四烯酰基残基的二-花生四烯酸-PE(di-A-PE)的过氧化作用而发生 在sn-1和sn-2两个位置。初步数据显示，15LOX本身就很容易氧化di-A-PE，领先 开始出现铁性下垂。通常，di-A-PE用于合成内源性大麻素，特别是 花生四甲酰基酰胺(双乙酰胺)。该途径利用di-A-PE的sn-1酰基进行酶促转酰化。 另一种PE的氨基，生成N-酰基-PE(NAPE)。在铁性下垂的情况下，N-转酰化 是15-LOX氧化di-A-PE的有力竞争者。因此，需要ACSL4的di-AA-PE合成可能是 上睑下垂的瓶颈。我们的中心假设是：i)脑外伤引起的下垂是一个两阶段的过程 由二-A-PE过氧化引发，然后是单一A-PE物种被氧化的繁殖阶段 在较低的比率下；ii)与NAPE/ANANDAME相比，di-A-PE的形成是铁性下垂敏感性的决定性因素 颅脑损伤后形成。我们的具体目标包括：1.优化分子的SIMS成像方案 正常和损伤脑中不同细胞类型的plx及其底物的特征。2.确定 ACSL4和NAPE在不同类型损伤细胞脂质过氧化的分子特异性中的作用 大脑。3.利用单细胞SIMS成像评价基于新机制的选择性治疗的有效性 铁下垂抑制剂：1)双烯丙基去氢花生四烯酸，2)15LOX/PEBP1复合体的抑制剂。

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.