Chronic alcohol-induced mitochondrial oxidant stress and Alzheimer's related pathogenesis

项目摘要 酒精是最常见的滥用物质，阿尔茨海默病（AD）是最常见的 神经退行性疾病酒精滥用是AD发展的重要风险因素， 酒精滥用和AD的交叉呈现出巨大的公共卫生问题。长期大量饮酒 与AD发病风险较高和疾病进展加速相关。临床 AD阶段之前是长达数十年的临床前阶段，其特征在于 淀粉样蛋白B（Ab）和蓝斑（LC）（脑干中的去甲肾上腺素核）中的神经元损失。我们 提出了一种新的机制，慢性酒精消耗使LC神经元容易受到 退化我们最近发现了一种新的细胞机制， 轴突中的氧化应激胞浆单胺，包括去甲肾上腺素，被单胺代谢 氧化酶（MAO）酶和从这个过程中产生的电子直接穿梭到 线粒体膜间隙这会选择性地增加线粒体氧化应激， 轴突我们认为，长期饮酒激活了轴突线粒体的这种新机制， 氧化应激导致LC神经元的退行性级联反应。我们的初步研究表明， 间歇性饮酒降低VMAT 2 mRNA，增加LC轴突氧化应激。 重要的是，我们还表明，在经历慢性， 间歇性饮酒与年龄匹配的饮水小鼠相比。因此，我们假设， 慢性间歇性饮酒降低了LC中VMAT 2的表达，导致LC中VMAT 2的表达增加。 在一些实施方案中，细胞质去甲肾上腺素通过MAO代谢，其然后引起轴突线粒体氧化应激。这 升高的线粒体氧化应激则会加速小鼠LC变性和Ab沉积 AD模型我们将使用2种AD遗传小鼠模型，APP/PS1和APP-NL敲除来检验这一假设。 在小鼠中，其发展进行性Ab病理学。结合尖端的双光子激光扫描 离体脑切片显微镜（2 PLSM），假荧光神经递质（FFN），遗传编码 将使用氧化还原生物传感器、免疫组织化学、体视学、药理学和遗传学技术 为了研究慢性间歇性饮酒对VMAT 2表达的影响， 包装去甲肾上腺素，LC轴突线粒体氧化应激，LC变性，Ab病理学，和 MAO依赖性。在目标1中，我们将确定慢性间歇性饮酒对LC的影响 轴突线粒体氧化应激在目标2中，我们将确定长期饮酒对 LC变性和Ab病理学。我们提出的实验将是第一个探索一种新的轴突 线粒体氧化应激介导的神经退行性变机制 慢性酒精消耗和AD。

PROJECT SUMMARY Alcohol is the most commonly abused substance and Alzheimer’s disease (AD) is the most common neurodegenerative disease. Alcohol abuse is a significant risk factor for the development of AD and this intersection of alcohol abuse and AD presents an enormous public health concern. Chronic, heavy alcohol use is associated with a higher risk of developing AD and accelerated progression of the disease. The clinical phase of AD is preceded by a decades long preclinical phase that is characterized by early deposition of amyloid b (Ab) and neuronal loss in the locus coeruleus (LC), a norepinephrine nucleus in the brainstem. We propose a novel mechanism by which chronic alcohol consumption renders LC neurons vulnerable to degeneration. We recently discovered a novel cellular mechanism that selectively induces mitochondrial oxidant stress in axons. Cytosolic monoamines, including norepinephrine, are metabolized by monoamine oxidase (MAO) enzymes and the electrons generated from this process are directly shuttled into the mitochondrial intermembrane space. This produces increased mitochondrial oxidant stress selectively in axons. We propose that chronic alcohol consumption activates this novel mechanism of axonal mitochondrial oxidant stress leading to a degenerative cascade in LC neurons. Our pilot studies suggest that chronic, intermittent alcohol consumption decreased VMAT2 mRNA and increased axonal oxidant stress in the LC. Importantly, we also showed that Ab pathology was increased in APP/PS1 mice that underwent chronic, intermittent alcohol consumption compared to age-matched water drinking mice. Thus, we hypothesize that chronic, intermittent alcohol consumption decreases VMAT2 expression in the LC leading to increased metabolism of cytosolic norepinephrine by MAO, which then causes axonal mitochondrial oxidant stress. This elevated mitochondrial oxidant stress would then accelerate LC degeneration and Ab deposition in mouse models of AD. We will test this hypothesis using 2 genetic mouse models of AD, APP/PS1 and APP-NL knock- in mice, which develop progressive Ab pathology. A combination of cutting-edge two-photon laser scanning microscopy (2PLSM) in ex vivo brain slices, false fluorescent neurotransmitters (FFNs), genetically encoded redox biosensors, immunohistochemistry, stereological, pharmacological, and genetic techniques will be used to investigate the effects of chronic, intermittent alcohol consumption on VMAT2 expression, VMAT2 packaging of norepinephrine, LC axonal mitochondrial oxidant stress, LC degeneration, Ab pathology, and MAO-dependence. In aim 1, we will determine the effect of chronic, intermittent alcohol consumption on LC axonal mitochondrial oxidant stress. In aim 2, we will determine the effect of chronic alcohol consumption on LC degeneration and Ab pathology. Our proposed experiments will be the first to explore a novel axonal mitochondrial oxidant stress-mediated neurodegenerative mechanism underlying the interaction between chronic alcohol consumption and AD.