Vitamin E Neuroprotection: Novel Molecular Mechanisms

说明(申请人提供)：维生素E的神经保护特性是通过对α-生育酚的研究确定的，α-生育酚是维生素E家族的8个成员之一，大致被归类为生育酚和生育三烯醇。这一建议是基于一个引人注目的观察结果，即α-生育三烯醇，而不是α-生育酚，通过调节与其抗氧化特性无关的信号转导途径，在纳摩尔浓度下提供强大的神经保护。结果发现，在50-100 nM(补充人的血浆浓度的十分之一)下，α-生育三烯醇可防止谷氨酸诱导的培养的HT4细胞以及未成熟和成熟的原代皮质神经元的死亡。小鼠海马神经元细胞缺乏内在的兴奋性毒性途径，已被证实为表征谷氨酸氧化毒性的模型。用同型半胱氨酸或谷胱甘肽合成抑制剂丁硫氨酸亚磺胺攻击HT4或原代培养的大鼠胚胎皮质神经元时，也观察到α-生育三烯醇的神经保护作用。生育三烯醇是自然产生的，多年来一直被人类安全地食用，特别是在东南亚。我们观察到，喂养怀孕大鼠的棕榈油中所含的饮食中的α-生育三烯醇到达了母鼠的大脑。与母亲相比，胎儿大脑中这种形式的维生素E的可获得性要高出数倍。这项建议的目的是通过药理学、生物化学和遗传学方法来描述α-生育三烯醇发挥神经保护作用的机制。此外，维生素E的神经保护功能完全由其抗氧化性调节的教条将被重新审视。一个常用的标准是确定细胞反应系统中是否含有活性氧簇(ROS)，即该系统对抗氧化剂的敏感度。人们经常忽视的是，抗氧化剂分子可能具有强大的抗氧化剂非依赖性特性。我们的工作假设是，细胞外谷氨酸水平的升高触发了以下事件：i)pp60c-Src、细胞外信号调节激酶(ERK)和12-脂氧合酶(LOX)的激活，以及ii)耗尽[GSH]i导致高[ROS]i，高[Ca+]i，线粒体功能障碍最终导致神经元死亡。生育三烯醇在两个水平上控制这些反应：早期和晚期。在谷氨酸攻击12小时后，90%的细胞死亡的时间框架内，NM(50-250)α-生育三烯醇在谷氨酸处理的第一个小时内抑制可诱导的c-Src、ERK和12-LOX的激活构成了非抗氧化剂依赖的早期控制机制之一。[GSH]i的耗竭、[ROSJ]i的升高和随后的线粒体功能障碍都是紧密相连的，并由于其抗氧化特性而受到较高浓度的α-生育三烯醇(=或&gt；1微摩尔)的后期控制。这项研究将为在具有挑战性的条件下防止神经元死亡和确保神经元正常功能的安全和有效的方法奠定基础。

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.