Vitamin E Neuroprotection: Novel Molecular Mechanisms

描述（由申请人提供）：维生素E的神经保护特性已通过α-生育酚的研究确定，α-生育酚是维生素E家族的8个成员之一，广泛分类为生育酚和生育三烯酚。该提议是基于以下惊人的观察结果：α-生育三烯酚而不是α-生育酚通过调节信号转导途径而不依赖于其抗氧化性质，在纳摩尔浓度下赋予有效的神经保护作用。因此，发现在50-100 nM（补充人体血浆浓度的十分之一）时，α-生育三烯酚可防止培养的HT 4细胞以及未成熟和成熟的原代皮层神经元的谷氨酸诱导的死亡。小鼠HT海马神经元细胞缺乏内源性兴奋毒性通路，已被验证为表征氧化谷氨酸毒性的模型。神经保护作用的α-生育三烯酚时，也观察到同型半胱氨酸或丁硫氨酸亚砜亚胺（谷胱甘肽合成的抑制剂）被用来挑战HT 4或原代大鼠胎儿皮层神经元。生育三烯酚天然存在，多年来一直被人类安全食用，特别是在东南亚。我们观察到，喂食给怀孕大鼠的棕榈油中含有的膳食α-生育三烯酚到达母鼠的大脑。胎儿大脑中这种形式的维生素E的可用性比母亲高出数倍。本提案的目的是利用药理学、生物化学和遗传学方法来表征α-生育三烯酚发挥其神经保护作用的机制。此外，维生素E的神经保护功能仅由其抗氧化特性介导的教条将被重新审视。建立细胞应答系统中活性氧（ROS）参与的常用标准是该系统对抗氧化剂的敏感性。抗氧化剂分子可能具有有效的抗氧化剂非依赖性特性，这一点常常被忽视。我们的工作假设是细胞外谷氨酸水平升高触发以下事件：i）pp 60 c-Src、细胞外信号调节激酶（ERK）和12-脂氧合酶（LOX）的激活，和ii）耗尽[GSH]i，导致高[ROS]i、高[Ca 2 +]i、线粒体功能障碍，最终导致神经元死亡。生育三烯酚在两个层面控制这些反应：早期和晚期。在谷氨酸激发12小时后> 90%的细胞死亡的时间范围内，通过nM（50-250）α-生育三烯酚在谷氨酸处理的前1小时内抑制诱导型c-Src、ERK和12-LOX活化构成了抗氧化剂非依赖性的早期控制机制之一。[GSH]i的消耗、[ROSJ]i的升高和随后的线粒体功能障碍都是紧密相关的，并且由于其抗氧化性质而受到较高（=或> 1微摩尔）浓度的α-生育三烯酚的后期控制。这项研究将为一种安全有效的方法奠定基础，以防止神经元在具有挑战性的条件下死亡并确保其正常功能。

DESCRIPTION (provided by applicant): Neuroprotective properties of vitamin E have been established from the study of alpha-tocopherol, one of 8 members of the vitamin E family broadly categorized as tocopherols and tocotrienols. This proposal is based on a striking observation that alpha-tocotrienol, but not aipha-tocopherol, confers potent neuroprotection at nanomolar concentrations through regulation of signal transduction pathways independent of its antioxidant property. Thus, it was found that at 50-100 nM (one-tenth of plasma-concentration in supplemented humans) alpha-tocotrienol prevented glutamate-induced death of cultured HT4 cells as well as that of both immature and mature primary cortical neurons. Murine HT hippocampal neuronal cells, lacking intrinsic excitotoxicity-pathway, have been validated as a model to characterize oxidative glutamate toxicity. Neuroprotective effects of alpha-tocotrienol were also observed when homocysteic acid or buthionine sulfoximine (inhibitor of GSH synthesis) was used to challenge HT4 or primary rat fetal cortical neurons. Tocotrienols occur naturally and has been safely consumed by humans, particularly in Southeast Asia, for years. We observed that dietary alpha-tocotrienol contained in palm oil fed to pregnant rats reaches the brain of mother rats. Availability of this form of vitamin E is multi-fold higher in fetal brain compared to that of the mother. The objective of this proposal is to characterize the mechanisms by which alpha-tocotrienol exerts its neuroprotective action using pharmacological, biochemical and genetic approaches. In addition, the dogma that the neuroprotective function of vitamin E is solely mediated by its antioxidant property will be revisited. A commonly used criterion to establish the involvement of reactive oxygen species (ROS) in a cell-response system is sensitivity of that system to antioxidants. It is often ignored that antioxidant molecules may have potent antioxidant-independent properties. Our working hypothesis is that elevated levels of extracellular glutamate trigger the following events: i) activation of pp6O c-Src, extracellular signal regulated kinase (ERK) and 12-lipoxygenase (LOX), and ii) depletes [GSH]i resulting in high [ROS]i, high [Ca2+]i, mitochondrial dysfunction eventually causing neuronal death. Tocotrienol controls these responses at two levels: early and late. In a time frame where >90 percent of the cells are dead after 12h of glutamate challenge, inhibition of inducible c-Src, ERK and 12-LOX activation within the first 1 h of glutamate treatment by nM (50-250) alpha-tocotrienol constitutes one of the early control mechanisms that is antioxidant-independent. Depletion of [GSH]i, elevation of [ROSJ]i and subsequent mitochondrial dysfunction are all tightly linked and are subject to late-control by higher (= or> 1 micromolar) concentrations of alpha-tocotrienol by virtue of its antioxidant property. This study will lay the foundation for a safe and efficient approach to prevent death and secure normal functioning of neurons under challenging conditions.