Vitamin E Neuroprotection: Novel Molecular Mechanisms

描述（由申请人提供）：维生素E的神经保护特性已经从α -生育酚的研究中确立，α -生育酚是维生素E家族的8个成员之一，被广泛归类为生育酚和生育三烯醇。这一建议是基于一项惊人的观察，即-生育三烯醇，而不是-生育酚，在纳摩尔浓度下通过调节独立于其抗氧化特性的信号转导途径，赋予有效的神经保护作用。因此，研究发现，在50-100 nM（补充人血浆浓度的十分之一）下，α -生育三烯醇可以防止谷氨酸诱导的HT4细胞以及未成熟和成熟的初级皮质神经元的死亡。缺乏内在兴奋毒性通路的小鼠HT海马神经元细胞已被证实为表征谷氨酸氧化毒性的模型。当用同型半胱酸或丁硫氨酸亚砜胺（GSH合成抑制剂）刺激HT4或原代大鼠胎儿皮质神经元时，也观察到α -生育三烯醇的神经保护作用。生育三烯醇天然存在，多年来一直被人类安全食用，特别是在东南亚。我们观察到，喂给怀孕大鼠的棕榈油中含有的α -生育三烯醇到达了母鼠的大脑。胎儿大脑中这种维生素E的含量是母体的数倍。本提案的目的是表征-生育三烯醇发挥其神经保护作用的机制，使用药理学，生化和遗传学方法。此外，关于维生素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。确定活性氧（ROS）参与细胞反应系统的一个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强大的不依赖于抗氧化剂的特性，这一点经常被忽视。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)激活pp60 c-Src，细胞外信号调节激酶（ERK）和12-脂氧合酶（LOX）， ii)消耗[GSH]i导致高[ROS]i，高[Ca2+]i，线粒体功能障碍最终导致神经元死亡。生育三烯醇在两个层面上控制这些反应：早期和晚期。在谷氨酸刺激12小时后，90%的细胞死亡，nM (50-250) α -生育三烯醇在谷氨酸处理的前1小时内抑制诱导的c-Src、ERK和12-LOX的激活，构成了不依赖抗氧化剂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α -生育三烯醇的抗氧化特性，它们受到较高（=或bb0.1微摩尔）浓度的后期控制。该研究将为在具有挑战性的条件下安全有效地预防死亡和确保神经元正常功能奠定基础。

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.