Removing copper for pennies: an affordable strategy for Alzheimer's

描述(由申请人提供)： 目的：总体目标是评估铜螯合策略预防和治疗阿尔茨海默病的可行性。具体目的：1)确定在AD病理出现之前开始铜锌络合治疗是否是一种有效的病理修饰疗法。2)确定在AD病理发生后开始补锌是否有效。3)探讨铜负荷是否加速AD病理的出现。计划：口服硫酸锌将实现铜的络合作用，这是一种在动物模型和人类患者中公认的安全干预措施。该方法是基于一种更有效的铜络合剂(四硫代钼酸盐)的经验，这种络合剂可能毒性太高，不能在人体内使用。方法：所有实验均采用转基因AD小鼠模型(Tg2576和Laferla三重转基因小鼠)。锌和铜将按照既定的方案在饮用水中进行添加。铜的去除将通过监测血浆铜蓝蛋白来滴定。治疗结束后，用Morris水迷宫和大脑进行空间记忆评估，然后采集大脑并进行分割。可溶和不溶淀粉样蛋白的前脑水平将通过酶联免疫吸附试验和免疫组织化学方法测定，寡聚体β淀粉样蛋白的水平通过蛋白质印迹测定，铜的水平通过原子吸收光谱测定。其他感兴趣的蛋白质将通过Western印迹来测量，感兴趣的基因的表达将通过定量RT-PCR来测量。迄今为止的发现：1)在阿尔茨海默病转基因小鼠模型中，当四硫钼酸盐(TM)在通常出现病理的年龄之前开始铜络合治疗时，大脑中的铜和不溶性β淀粉样蛋白减少。2)TM处理不影响脑内铜依赖酶的活性。3)TM治疗几个月后对小鼠的行为功能无损害。4)与TM相比，锌处理可降低血浆铜蓝蛋白和脑铜含量，降低过度螯合风险。临床相关性：这些数据将用于设计预防人类AD的临床试验。 公共卫生相关性： 这项建议涉及一个非常重要的退伍军人医疗保健问题，即阿尔茨海默病的预防和治疗。阿尔茨海默氏症是晚年残疾的主要来源，并困扰着大量退伍军人。这里提出的实验是用小鼠来测试旨在降低大脑铜水平的治疗方法是否会降低阿尔茨海默病的风险，或许还会减缓阿尔茨海默病的进展速度。这些研究是基于“试管”实验，这些实验表明铜可能会与脑组织相互作用，促进阿尔茨海默病中发生的一些变化。尽管这些“试管”研究令人信服，但除非在动物模型中证实这些结果，否则不可能在人类受试者身上提出这种类型的治疗方法，因为动物模型更接近于人类的情况。我们建议使用特殊的小鼠，这些小鼠被设计成在大脑中产生类似阿尔茨海默氏症的变化。这些小鼠将接受一种简单、安全的治疗，降低大脑中的铜水平。然后将检查小鼠的记忆和大脑生化，以确定治疗是否达到了预期的效果。如果它如预期的那样起作用，我们将建议对有阿尔茨海默病风险的人类受试者进行进一步研究。如果它不起作用，我们将试图理解为什么不能，以便为包括老年退伍军人在内的人类患者制定更好的治疗策略。

DESCRIPTION (provided by applicant): Objectives: The overall objective is to evaluate the feasibility of copper chelating strategies for the prevention and treatment of Alzheimer's disease. Specific aims: 1) To determine if copper complexing with zinc is an effective pathology-modifying therapy if initiated prior to the appearance of AD pathology. 2) To determine if zinc is effective if initiated after the onset of AD pathology. 3) To determine if copper loading accelerates the appearance of AD pathology. Plan: Copper complexing will be achieved with oral zinc sulfate, an established and safe intervention in animal models and human patients. The approach is based on experience with a more potent copper complexing agent (tetrathiomolybdate) which may be too toxic for use in human subjects. Methods: Transgenic mouse models of AD (Tg2576 and the Laferla triple transgenic mice) will be used for all of these experiments. Zinc and copper will be administered in the drinking water according to established protocols. Copper removal will be titrated by monitoring of plasma ceruloplasmin. At the end of the treatment period, spatial memory will be assessed by Morris Water Maze and brains then harvested and divided. Forebrain levels of soluble and insoluble amyloid species will be determined by ELISA and immunohistochemistry; oligomeric beta amyloid levels by western blot, copper levels by atomic absorption spectroscopy. Other proteins of interest will be measured by Western blot, and the expression of genes of interest by quantitative RT-PCR. Findings to date: 1) Brain copper and insoluble beta amyloid are reduced in a transgenic mouse model of AD when copper complexing therapy is initiated with tetrathiomolybdate (TM) prior to the age at which pathology typically appears. 2) TM treatment does not impair brain activity of copper dependent enzymes. 3) TM treatment does not impair behavioral function in mice treated for several months. 4) Zinc treatment lowers plasma ceruloplasmin and brain copper with less risk of over- chelation compared to TM. Clinical relevance: These data will be used to design clinical trials for prevention of AD in human subjects. PUBLIC HEALTH RELEVANCE: This proposal is relevant to a very important veterans' health care issue, namely the prevention and treatment of Alzheimer's disease. Alzheimer's disease is a major source of disability in late life, and afflicts vast numbers of veterans. The experiments proposed here use mice to test the possibility that therapies directed at lowering brain levels of copper will reduce the risk and perhaps slow the rate of progression of Alzheimer's disease. These studies are based on "test-tube" experiments which suggest that copper may interact with brain tissue to promote some of the changes that occur in Alzheimer's disease. Although these "test-tube" studies are convincing, it will not be possible to propose this type of treatment in human subjects unless there is confirmation of these results in an animal model, which more closely approximates the situation in human beings. We propose to use special mice which have been engineered to develop Alzheimer's-like changes in the brain. The mice will be treated with a simple, safe treatment which lowers brain levels of copper. The mouse memory and brain biochemistry will then be examined to determine if the treatment had its intended effect. If it works as expected, we will propose further studies in human subjects at risk of Alzheimer's disease. If it does not work, we will attempt to understand why not, in order to develop better treatment strategies for human patients, including aging veterans.