Oxidative Lipidomics in Pediatric Traumatic Brain Injury

在美国，每年儿童严重TBI导致17400例死亡和60000例住院治疗。百分之五十 存活的严重TBI儿童在6个月时的神经学结果较差。儿童严重TBI是 因此是迫切需要有效治疗的关键问题。自由基和氧化应激一直是 一致接受为TBI的普遍致病机制，促使抗氧化剂的治疗用途。 然而，非特异性自由基清除剂/抗氧化剂的临床试验失败了。这表明， TBI氧化还原失衡的来源和机制尚未确定，代表了一种潜在的治疗方法， 机会在之前的资助期间，我们发现未成熟大脑中TBI后的脂质过氧化作用 作为受控酶促反应的结果发生。我们发现线粒体的过氧化作用 磷脂心磷脂（CL）代表了出生后TBI后神经元凋亡的必要阶段 (PND)17只老鼠我们确定了细胞色素c作为CL过氧化反应的催化剂，并表明线粒体- CL过氧化的靶向抑制剂抑制TBI诱导的细胞凋亡，并保护认知功能。 PND 17大鼠。我们的最新工作确定了高选择性氧化花生四烯酸（AA）， 磷脂酰乙醇胺（PE）通过15脂氧合酶（15 LOX）导致铁凋亡。我们发现 PE结合蛋白1（PEBP 1）与15 LOX复合，并将其底物特异性从游离AA改变为 AA酯化成PE以产生过氧化氢-AA-PE死亡信号。通常过氧化氢-AA-PE是 通过谷胱甘肽过氧化物酶4（GPX 4）/谷胱甘肽（GSH）的联合作用消除。不成熟的大脑 因此，与成人脑相比，较低的GSH水平和GPX活性可能更容易在损伤时发生铁凋亡。 事实上，我们的初步数据表明，TBI导致15 LOX的表达和活性显著增加， PND 17大鼠脑中氧化AA-PE的蓄积。此外，初步数据表明， AA-PE氧化抑制TBI诱导的神经元死亡并保护认知功能。因此我们 假设通过15 LOX/PEBP 1复合物产生氧化AA-PE导致神经元死亡， 代表了药物发现的新目标，导致儿科TBI的创新疗法。我们建议测试 我们的假设在三个具体目标。目标1将确定环境影响的程度、空间和时间模式， 15 TBI后LOX/PEBP 1复合物形成和AA-PE氧化。目的2将研究 TBI诱导的神经元死亡中的AA-PE氧化。目标3将设计和研究作用机制， TBI中15 LOX和15 LOX/PEBP 1活性的小分子调节剂的神经保护潜力。这些 研究将采用强大的脂质组学和氧化脂质组学技术，提供重要的机制， 儿童TBI后PE氧化在神经元铁凋亡中的作用。能够选择性 调节PE氧化，铁凋亡机制中的关键早期事件，可能导致靶向治疗 并最终改善脑损伤后儿童的预后。

Each year in the US, severe TBI in children results in ∼7400 deaths and 60 000 hospitalizations. Fifty percent of surviving children with severe TBI have poor neurological outcome at six months. Severe TBI in children is thus a critical problem in desperate need of impactful therapies. Free radicals and oxidative stress have been uniformly accepted as universal pathogenic mechanisms of TBI prompting therapeutic use of antioxidants. Invariably, clinical trials of non-specific free radical scavengers/antioxidants failed. This suggests that true sources and mechanisms of TBI redox disbalance remain undefined, and represent a potential therapeutic opportunity. During the previous funding period we showed that lipid peroxidation after TBI in immature brain occurs as a result of controlled enzymatic reactions. We discovered that peroxidation of mitochondrial phospholipid cardiolipin (CL) represents a required stage of neuronal apoptosis after TBI in postnatal day (PND) 17 rats. We identified cytochrome c as a catalyst of CL peroxidation and showed that a mitochondria- targeted inhibitor of CL peroxidation suppressed TBI-induced apoptosis and preserved cognitive function in PND17 rats. Our latest work identified highly selective oxidation of arachidonic acid (AA) containing phosphatidylethanolamines (PE) by 15 lipoxygenase (15LOX) to be causative to ferroptosis. We discovered that PE binding protein 1 (PEBP1) complexes with 15LOX and changes its substrate specificity from free AA to AA esterified into PE to generate hydroperoxy-AA-PE death signals. Normally hydroperoxy-AA-PE are eliminated by combined action of glutathione peroxidase 4 (GPX4)/glutathione (GSH). Immature brain has lower GSH levels and GPX activity vs adult brain thus could be more vulnerable to ferroptosis upon injury. Indeed our preliminary data show that TBI leads to marked increase in expression and activity of 15LOX, and accumulation of oxidized AA-PE in PND17 rat brain. Furthermore, preliminary data indicate that inhibition of AA-PE oxidation suppresses TBI-induced neuronal death and preserves cognitive function. Thus, we hypothesize that generation of oxidized AA-PE by 15LOX/PEBP1 complex leads to neuronal death and represents a new target for drug discovery leading to innovative therapies in pediatric TBI. We propose to test our hypothesis in three Specific Aims. Aim 1 will determine the degree, spatial and temporal pattern of 15LOX/PEBP1 complex formation and AA-PE oxidation after TBI. Aim 2 will investigate the mechanisms of AA-PE oxidation in TBI-induced neuronal death. Aim 3 will design and investigate the mechanism of action and neuroprotective potential of small-molecule regulators of 15LOX and 15LOX/PEBP1 activity in TBI. These studies will employ powerful lipidomics and oxidative lipidomics technology to provide important mechanistic information on the role of PE oxidation in neuronal ferroptosis after pediatric TBI. The ability to selectively modulate PE oxidation, a critical early event in the mechanism of ferroptosis, could lead to targeted therapies for TBI and ultimately improve outcome for children after brain injury.