Vitamin C Transporters in the Brain

大脑中的维生素 C 转运蛋白

• 批准号: 7860690
• 负责人: JAMES M. MAY
• 金额: $ 29.92万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7860690
• 关键词: Affect; Affinity Labels; Alzheimer' s Disease; Animals; Antioxidants; Ascorbic Acid; Birth; Brain; Cells; Cerebral hemisphere hemorrhage; Cessation of life; Clinical Research; Cysteine; Development; Electrophysiology (science); Engineering; Glutamates; Goals; Homeostasis; Huntington Disease; In Vitro; Infarction; Injury; Knock-out; Knockout Mice; Membrane Proteins; Modeling; Mus; Mutagenesis; Myelin; Nervous System Physiology; Neuraxis; Neuroblastoma; Neurodegenerative Disorders; Neurologic; Neurons; Oxidants; Oxidation-Reduction; Parkinson Disease; Peptides; Proteins; Regulation; Reperfusion Injury; Resistance; Role; Stroke; Structure-Activity Relationship; Synapses; Testing; Tissues; Toxic effect; Transgenic Mice; Transgenic Organisms; Vitamin C in Brain Pathway; Vitamin E Deficiency; Vitamins; Xenopus oocyte; affinity labeling; amidation; ascorbate; extracellular; human disease; in vivo; novel; oxidant stress; prevent; public health relevance; response; sodium-dependent vitamin C transporter 2

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.

描述(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因氧化应激增加而引起的并发症。正常情况下，抗坏血酸在大脑中保持相对较高的浓度，特别是在神经元中，它作为一种抗氧化剂，支持几个重要的神经元功能。低毫摩尔范围内的抗坏血酸浓度是由一种称为SVCT2的特定抗坏血酸转运蛋白在神经元中产生的。缺乏这种转运蛋白的转基因小鼠的脑部抗坏血素含量非常低，出生时就死于脑出血，这表明维生素对生存至关重要。这项提议的总体目标是确定SVCT2转运体在神经元中是如何调节的，以及转运体数量的变化是否会在体外和体内影响神经元的功能和抗氧化防御。有三个具体目标。在第一个目标中，将在培养的SH-SY5Y神经母细胞瘤细胞和非洲爪哇卵母细胞中评估SVCT2的调节机制和结构-功能关系。在SH-SY5Y细胞中，将评估氧化应激对SVCT2表达和活性的调节作用。此外，将通过SH-SY5Y细胞中的亲和标记和非洲爪哇卵母细胞中的突变方法来评估关键半胱氨酸在蛋白质胞外表面的功能作用。在第二个目标中，将确定缺乏SVCT2基因的敲除小鼠在出生时死亡的机制。同样在目标2中，由于维生素E和C的联合缺乏而导致的氧化应激的新模型将被用来确定与表达正常或降低(50%)SVCT2的小鼠相比，表达增加的SVCT2的转基因小鼠是否受到神经损伤的保护。在第三个目标中，将从表达几种不同水平SVCT2(转基因、野生型、杂合敲除和纯合子敲除)的小鼠中制备原代培养神经元。这些细胞将被用来测试SVCT2的表达是否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力，3)促进培养的皮质神经元的成熟，以及3)影响神经元的电生理。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的功能、发育和生存至关重要。公共卫生相关性：该项目研究大脑中维生素C转运体的功能、调节和组织表达，与神经元功能、成熟和对氧化应激的抵抗力相关。包括阿尔茨海默氏症和帕金森氏症在内的几种神经退行性疾病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。