amyloid-Beta and Altered Cellular Metabolism in Alzheimer's Disease

 描述(申请人提供)：阿尔茨海默病(AD)是一种无法治愈的神经退行性疾病，影响着500多万美国人。病理上，由淀粉样β蛋白(A？)组成的细胞外斑块和由tau蛋白组成的细胞内缠结是阿尔茨海默病的特征。阿尔茨海默病的病因不明。然而，患者的早期变化表明，受影响区域的葡萄糖代谢发生了变化。具体地说，葡萄糖优先通过糖酵解为乳酸或通过磷酸戊糖途径代谢，而不是继续氧化磷酸化，当有氧存在时，这一过程称为有氧糖酵解。先前在细胞系和原代培养中的研究进一步表明，A？导致 有氧糖酵解增加。从生理上讲，神经元的糖酵解率很低，糖酵解率增加会导致细胞凋亡。这表明AD患者有氧糖酵解增加可能导致神经元功能障碍和毒性。我们假设Aü导致有氧糖酵解增加，这有助于神经元功能障碍和AD的进展。由于之前的许多研究没有区分神经元和神经胶质的贡献，也没有研究突触代谢变化的影响，我们正在使用能够进行精确定位测量的工具。在目标1中，我们将开发一种使用基于FRET的葡萄糖传感器来测量亚细胞分辨率的有氧糖酵解的方法，并通过质谱仪与充分表征的代谢跟踪进行验证。我们的初步数据表明，我们可以监测单个突触、神经元胞体和神经胶质细胞中的葡萄糖水平。一旦我们优化了我们的测试，我们将测量阿伯的S对细胞体和突触神经元葡萄糖代谢的影响。通过从药理上控制葡萄糖的摄取和消耗，我们将通过有氧糖酵解和其他代谢途径来量化葡萄糖的流量，以确定Aü如何改变代谢。在目标2中，我们将深入研究改变的有氧糖酵解的生物能量效应，以确定代谢变化如何影响神经元的功能。我们的实验室已经开发出一种方法来测量突触的ATP水平。该检测使用基于FRET的ATP传感器来区分线粒体和糖酵解来源的ATP。我们将使用vGlut1-pHluorin构建来测试A？对ATP水平的影响，以及在功能上对突触传递的影响。在目标3中，我们将在体内工作，以确定有氧糖酵解是否在AD小鼠模型中发生变化，以及不同的细胞代谢如何改变AD中的神经元功能。我们将使用核磁共振波谱来测试AD小鼠模型是否显示出有氧糖酵解增加。然后，我们将通过敲除海马区的PKM1来增加AD小鼠模型中的有氧糖酵解。我们将通过行为和病理评估来测试增加的有氧糖酵解是否有害。在这个项目完成后，我们将确定A？是否增加有氧糖酵解，以及这如何在AD模型中促进毒性，测试潜在的治疗靶点。我们还将开发一种在单细胞水平上测量葡萄糖代谢的方法。

 DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is an incurable neurodegenerative disease affecting more than 5 million Americans. Pathologically, extracellular plaques made of the protein amyloid-beta (Aß) and intracellular tangles made of the protein tau characterize AD. The cause of AD is unknown. However, early changes in patients show that glucose metabolism is altered in affected areas. Specifically, glucose is metabolized preferentially through glycolysis to lactate or through the pentose phosphate pathway rather than continuing on to oxidative phosphorylation, a process termed aerobic glycolysis when oxygen is present. Prior work in cell lines and primary culture further indicates that Aß leads to increased aerobic glycolysis. Physiologically, neurons have low glycolytic rates and an increase causes apoptosis. This suggests that increased aerobic glycolysis in AD may contribute to neuronal dysfunction and toxicity seen in the disease. We hypothesize that Aß causes increased aerobic glycolysis, and that this contributes to neuronal dysfunction and the progression of AD. Because many previous studies did not differentiate between neuronal and glial contributions, nor did they examine the effect of metabolic changes at the synapse, we are using tools that allow precise localized measurements. In Aim 1, we will develop an assay to measure aerobic glycolysis with subcellular resolution using a FRET-based glucose sensor, and validated against well-characterized metabolic tracking using mass spectrometry. Our preliminary data demonstrate that we can monitor glucose levels in individual synapses, neuronal cell bodies and glia. Once we have optimized our assay, we will measure Aß's effect on glucose metabolism in neurons at the cell body and synapse. By controlling glucose uptake and consumption pharmacologically, we will quantify the flux of glucose through aerobic glycolysis and other metabolic pathways to determine how Aß changes metabolism. In Aim 2, we will delve into the bioenergetic effect of altered aerobic glycolysis to determine how metabolic changes functionally impact neurons. Our lab has developed an assay to measure ATP levels at the synapse. The assay differentiates between mitochondrial and glycolytic- derived ATP using a FRET-based ATP sensor. We will test the effect of Aß on ATP levels and, functionally, its effect on synaptic transmission with a vGlut1-pHluorin construct. In Aim 3, we will work in vivo to establish whether aerobic glycolysis changes in an AD mouse model and how varying cellular metabolism modifies neuronal function in AD. We will test whether an AD mouse model shows increased aerobic glycolysis using nuclear magnetic resonance spectroscopy. We will then increase aerobic glycolysis in an AD mouse model by knocking out PKM1 in the hippocampus. We will test if increased aerobic glycolysis is detrimental, through behavioral and pathological evaluation. Upon completion of this project, we will have determined whether Aß increases aerobic glycolysis and how this contributes to toxicity in models of AD, testing a potential therapeutic target. We will have also developed a method to measure glucose metabolism on a single cell level.