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集中在阐明金属引起或与阿尔茨海默病(AD)和威尔逊病(WD)等神经系统疾病相关的机制。这项建议中描述的实验将侧重于铜在WD中的作用，WD是一种铜代谢的遗传性疾病，与严重的肝脏、神经和精神异常有关。虽然WD的特征是肝脏铜蓄积导致暴发性肝炎和/或脑铜蓄积导致严重的神经症状，但累积铜引发器官或细胞水平的分子变化的机制尚不是很清楚。我们建议的中心假设是，积累的肝脏铜会导致肝细胞氧化还原电位的变化，最终将细胞推向凋亡或坏死。我们将在4个具体目标上测试我们的假设，使用针对哺乳动物组织定制的光谱方法和传统生化方法的组合。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相结合，我们将确定对照组小鼠和WD动物模型ATP7B-/-小鼠在关键疾病阶段(特定目标1)的肝组织中细胞和细胞内铜的浓度、分布和氧化状态。然后，我们将使用分离的匀浆和SXRF/荧光显微镜(特异性AIM2)相结合的方法，确定铜积累的细胞内位置(细胞器)，并确定可以在ATP7B-/-小鼠的肝组织中作为铜结合蛋白的候选蛋白。细胞模型(HepG2和MC65细胞)中三个中心的硫醇/二硫化物氧化还原对(GSH/GSSG，Trx1(SH)2/SS，Cys/CySS)的氧化还原电位将在静息和高铜条件下测定(特定目标3)。结果将随后与不同年龄的ATP7B-/-和对照肝脏的氧化还原电位进行比较(特定目标4)。将我们在特定目标1和2中的发现与特定目标3和4中的结果相关联，将使我们能够表征铜积累对细胞氧化还原电位的影响，以及随后走向凋亡或坏死的周期的变化。 公共卫生相关性： 尽管组织中金属浓度或分布的变化是包括阿尔茨海默病和威尔逊病在内的各种人类疾病的标志，但其潜在的发病机制，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，铜在肝脏、大脑和肾脏中积聚。这项应用中提出的研究将全面研究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，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.