Vitamin C Transporters in the Brain

描述（由申请人提供）：动物和临床研究表明，维生素C或抗坏血酸有助于延缓或预防中风和几种神经退行性疾病中氧化应激增加引起的并发症。正常情况下，抗坏血酸在大脑中保持相对高浓度，尤其是在神经元中，它作为抗氧化剂并支持几种重要的神经元功能。在低毫摩尔范围内的抗坏血酸浓度是由一种特殊的抗坏血酸转运蛋白SVCT2在神经元中产生的。缺乏这种转运体的老鼠大脑中抗坏血酸含量非常低，出生时死于脑出血，这表明维生素对生存至关重要。本提案的总体目标是确定SVCT2转运体在神经元中的调节方式，以及转运体数量的变化是否会影响神经元功能和抗氧化防御，无论是在体外还是在体内。有三个具体目标。在第一个目的中，我们将在培养的SH-SY5Y神经母细胞瘤细胞和爪蟾卵母细胞中评估SVCT2的调控机制和结构-功能关系。我们将在SH-SY5Y细胞中评估SVCT2表达和活性对氧化应激反应的调节。此外，将通过SH- SY5Y细胞的亲和标记和非洲爪蟾卵母细胞的诱变方法来评估关键半胱氨酸在该蛋白细胞外表面的功能作用。在第二个目标中，将确定缺乏SVCT2的敲除小鼠出生时死亡的机制。同样在Aim 2中，一个由于维生素E和C联合缺乏而引起的氧化应激的新模型将被用来确定与表达正常或减少（50%）SVCT2的小鼠相比，表达增加SVCT2量的转基因小鼠是否免受神经损伤。在第三个目标中，原代培养神经元将从表达几种不同水平的SVCT2（转基因、野生型、杂合敲除和纯合敲除）的小鼠中制备。这些细胞将用于测试SVCT2表达是否1)决定神经元维持细胞内抗坏血酸的能力，2)增加神经元对氧化应激的抵抗能力，3)促进皮层神经元在培养中的成熟，以及3)影响神经元电生理。这些研究的结果有望表明，受氧化应激调节的SVCT2抗坏血酸转运体对神经元功能、发育和存活至关重要。公共卫生相关性：本项目研究大脑中维生素C转运体的功能、调控和组织表达，与神经元功能、成熟和抗氧化应激有关。包括阿尔茨海默病和帕金森病在内的几种神经退行性疾病都与氧化应激增加有关，因此该项目将有助于确定维生素C在大脑氧化损伤中的作用。

DESCRIPTION (provided by applicant): Vitamin C, or ascorbic acid, has been shown in animal and clinical studies to help delay or prevent complications attributed to increased oxidant stress in stroke and in several neurodegenerative disorders. Normally, ascorbate is maintained at relatively high concentrations in brain and especially in neurons, where it serves as an antioxidant and supports several important neuronal functions. Ascorbate concentrations in the low millimolar range are generated in neurons by a specific ascorbate transporter, termed the SVCT2. Mice engineered to lack this transporter have very low brain ascorbate contents and die at birth with cerebral hemorrhage, indicating that the vitamin is crucial for survival. The overall goal of this proposal is to determine how the SVCT2 transporter is regulated in neurons, and whether changes in transporter number can affect neuronal function and antioxidant defenses, both in vitro and in vivo. There are three specific aims. In the first aim, mechanisms of SVCT2 regulation and structure-function relationships will be evaluated in cultured SH-SY5Y neuroblastoma cells and in Xenopus oocytes. Regulation of both SVCT2 expression and activity in response to oxidant stress will be assessed in SH-SY5Y cells. Further, the functional role of key cysteines on the extracellular surface of the protein will be evaluated by affinity labeling in SH- SY5Y cells and by mutagenesis approaches in Xenopus oocytes. In the second aim, the mechanism by which knock-out mice lacking the SVCT2 die at birth will be determined. Also in Aim 2, a novel model of oxidant stress due to combined deficiencies of vitamins E and C will be used to establish whether transgenic mice expressing increased amounts of SVCT2 are protected from neurologic damage compared to mice expressing normal or decreased (50%) SVCT2. In the third aim, primary culture neurons will be prepared from mice expressing several different levels of SVCT2 (transgenic, wild-type, heterozygous knock-out, and homozygous knock-out). These cells will be used to test whether SVCT2 expression 1) determines the ability of neurons to maintain intracellular ascorbate, 2) increases resistance of neurons to oxidant stress, 3) enhances maturation of cortical neurons in culture, and 3) affects neuronal electrophysiology. Results from these studies are expected to show that the SVCT2 ascorbate transporter, as regulated by oxidant stress, is crucial for neuronal function, development, and survival. PUBLIC HEALTH RELEVANCE: This project to study the function, regulation and tissue expression of the transporter for vitamin C in the brain has relevance for neuronal function, maturation and resistance to oxidant stress. Several neurodegenerative diseases including Alzheimer's and Parkinson's are associated with increased oxidant stress so that this project will help define the role of vitamin C in oxidant injury to the brain.