The Role of Hepatic Copper Accumulation in Wilson Disease

描述（由申请人提供）： 项目概述我们研究的长期目标是阐明金属引起或与神经系统疾病如阿尔茨海默病（AD）和威尔逊病（WD）相关的机制。本提案中描述的实验将集中于铜在WD中的作用，WD是一种与严重肝脏、神经和精神异常相关的铜代谢遗传性疾病。虽然WD的特征，肝铜蓄积导致暴发性肝炎和/或脑铜蓄积导致严重的神经系统症状，是已知的，积累的铜触发在器官或细胞水平的分子变化的机制不是很清楚。我们建议的中心假设是，积累肝铜导致肝细胞氧化还原电位的变化，最终推动细胞凋亡或坏死。我们将测试我们的假设在4个特定的目标，使用专门用于与哺乳动物组织和传统的生物化学方法的光谱方法的组合。使用基于同步加速器的X射线荧光结合基于金的双标记技术，我们将确定对照小鼠和ATP 7 b-/-小鼠（WD的动物模型）在关键疾病阶段的肝组织中的细胞和细胞内铜浓度、分布和氧化态（具体目标1）。然后，我们将确定铜积累的细胞内位点（细胞器），并使用分级匀浆和SXRF/荧光显微镜（特异性Aim 2）的组合，确定可能作为ATP 7 b-/-小鼠肝组织中铜结合蛋白的候选蛋白质。将在静息和高铜条件下测定细胞模型（HepG 2和MC 65细胞）中三种中心巯基/二硫键氧化还原对（GSH/GSSG、Trx 1（SH）2/SS、Cys/CySS）的氧化还原电位（具体目标3）。随后将结果与不同年龄的ATP 7 b-/-和对照肝脏的氧化还原电位进行比较（具体目标4）。我们从特定目标1和2的研究结果与特定目标3和4的相关性将使我们能够表征积累铜对细胞中氧化还原电位的影响以及随后细胞凋亡或坏死周期的变化。 公共卫生相关性： 尽管组织中金属浓度或分布的变化是各种人类疾病的标志，包括阿尔茨海默病和威尔逊病，但对潜在的疾病机制，特别是金属的作用，仍然知之甚少。威尔逊病是一种遗传性疾病，其中铜积累肝脏，大脑和肾脏。本申请中提出的研究将提供铜对细胞和细胞器中任何中心氧化还原对的氧化还原电位的影响的全面研究，并解释积累的铜是否会导致细胞凋亡或坏死。

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. PUBLIC HEALTH RELEVANCE: Although changes in concentration or distribution of metals in tissues are a hallmark of various human diseases, including Alzheimer's and Wilson disease, the underlying disease mechanisms, specifically the role of the metal, remain poorly understood. Wilson disease is a genetic disorder in which copper accumulates liver, brain and kidney. The proposed studies in this application will provide a comprehensive study of the effect of copper on the redox potential of any of the central redox couples in cells and organelles and explain whether accumulating copper can apoptosis or necrosis.