The Role of Hepatic Copper Accumulation in Wilson Disease

肝铜蓄积在威尔逊病中的作用

• 批准号: 8440350
• 负责人: Martina Ralle
• 金额: $ 26.85万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440350
• 关键词: Acinus organ component; Age; Alzheimer' s Disease; Animal Model; Antibodies; Apoptosis; Basal Ganglia; Biochemical; Biological Availability; Brain; Cell Death; Cell Line; Cell Nucleus; Cell model; Cells; Cerebrum; Control Animal; Copper; Couples; Cytoplasm; Data; Deposition; Development; Disease; Disulfides; Fluorescence; Fluorescence Microscopy; Fractionation; Glutathione Disulfide; Goals; Gold; Hepatic; Hepatic Tissue; Hepatitis; Hepatocyte; Hepatolenticular Degeneration; Hereditary Disease; Image; Immunohistochemistry; In Vitro; Kidney; Label; Lead; Literature; Liver; Mass Spectrum Analysis; Measurement; Metabolism; Metallothionein; Metals; Methods; Molecular; Monitor; Mus; Nature; Necrosis; Neurologic; Nuclear; Organ; Organelles; Oxidation-Reduction; Pathology; Patients; Proteins; Rest; Roentgen Rays; Role; Site; Spectrum Analysis; Staging; Stress; Sulfhydryl Compounds; Symptoms; Synchrotrons; Techniques; Testing; Time; Tissues; Western Blotting; absorption; base; cell type; copper-binding protein; effective therapy; gel electrophoresis; hepatocellular carcinoma cell line; human disease; insight; nervous system disorder; neuroblastoma cell; orexin A receptor; oxidation; prevent; public health relevance; research study; tool; uptake

DESCRIPTION (provided by applicant): Project Summary The long term goal of our studies are focused on the elucidation of mechanisms by which metals cause or are associated with neurological disorders such as Alzheimer's disease (AD) and Wilson disease (WD). This experiments described in this proposal will focus on the role of copper in WD, a genetic disorder of copper metabolism associated with severe hepatic, neurological, and psychiatric abnormalities. Although the hallmarks of WD, hepatic copper accumulation leading to fulminant hepatitis and/or cerebral copper accumulation leading to severe neurological symptoms, are known, the mechanisms by which accumulated copper triggers molecular changes at the organ or cellular levels is not very well understood. The central hypothesis of our proposal is that accumulating hepatic copper causes changes in the hepatocellular redox potential ultimately pushing the cells toward apoptosis or necrosis. We will test our hypothesis in 4 specific aims using a combination of spectroscopic approaches tailored to be used with mammalian tissue and traditional biochemical methods. Using synchrotron-based X-ray fluorescence in combination with a gold- based, dual labeling technique we will determine the cellular and intracellular copper concentration, distribution, and oxidation states in hepatic tissues of control mice and ATP7b-/- mice, an animal model for WD, at crucial disease stages (Specific Aim 1). We will then identify the intracellular sites (organelles) of copper accumulation and identify candidate proteins that could function as copper binding proteins in hepatic tissue of ATP7b-/- mice using fractionated homogenates and a combination of SXRF/fluorescence microscopy (Specific Aim2). Redox potentials in cellular models (HepG2 and MC65 cells) for the three central thiol/disulfide redox couples (GSH/GSSG, Trx1(SH)2/SS, Cys/CySS) will be determined under resting and high copper conditions (Specific Aim 3). The results will be subsequently compared to the redox potential of ATP7b-/- and control livers at different ages (Specific Aim 4). Correlation of our findings from Specific Aim 1 and 2 with those for Specific Aim 3 and 4 will allow us to characterize the effect of accumulating copper on the redox potential in cells and the subsequent changes in the cycle towards apoptosis or necrosis.

描述（由申请人提供）： 项目摘要我们的研究的长期目标集中在阐明金属引起或与阿尔茨海默氏病（AD）和威尔逊病（WD）等神经系统疾病相关的机制上。该提案中描述的这个实验将集中于铜在WD中的作用，铜在与严重的肝，神经和精神病异常相关的铜代谢遗传疾病中的作用。尽管WD的标志，肝铜的积累导致暴发性肝炎和/或脑铜的积累导致严重的神经系统症状，但已知的铜触发分子在器官或细胞水平上的分子变化的机制并不十分充分了解。我们建议的中心假设是，积累的肝铜会导致肝细胞氧化还原电位的变化，最终将细胞推向细胞凋亡或坏死。我们将使用适合哺乳动物组织和传统生化方法的光谱方法组合在4个特定目标中检验我们的假设。使用基于同步加速器的X射线荧光与基于金的双重标记技术结合使用，我们将确定对照小鼠的肝组织中的细胞和细胞内铜浓度，分布和氧化态在Contp7b - / - 小鼠，WD的动物模型，在关键疾病阶段（特定的AIM AIM 1）。然后，我们将鉴定铜积累的细胞内部位（细胞器），并鉴定候选蛋白，这些蛋白质可以用作ATP7B - / - 小鼠的肝组织中的铜结合蛋白，并使用SXRF/荧光显微镜组合（特定的AIM2）。在静止且高铜条件下（特定的AIM 3），将确定三个中央硫醇/二硫化物氧化还原夫妻（GSH/GSSG，TRX1（SH）2/SS，CYS/CYSS）的细胞模型（HEPG2和MC65细胞）中的氧化还原电位（GSH/GSSG，TRX1（SH）2/SS，CYS/CYSS）（特定的AIM 3）。随后将将结果与ATP7B - / - 和不同年龄的对照肝的氧化还原电位进行比较（特定AIM 4）。我们从特定目标1和2的发现与特定目标3和4的发现的相关性将使我们能够表征累积铜对细胞中氧化还原电位的影响，以及随后在凋亡或坏死的周期变化。