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

 描述（由申请人提供）：阿尔茨海默病（AD）是一种无法治愈的神经退行性疾病，影响超过500万美国人。在病理学上，由蛋白质淀粉样蛋白-β（A β）制成的细胞外斑块和由蛋白质tau制成的细胞内缠结表征AD。AD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改变。具体而言，葡萄糖优先通过糖酵解代谢为乳酸或通过戊糖磷酸途径代谢，而不是继续进行氧化磷酸化，当存在氧气时，该过程称为有氧糖酵解。先前在细胞系和原代培养中的工作进一步表明，Ablation导致 增加有氧糖酵解。在生理上，神经元具有低糖酵解速率，并且糖酵解速率的增加导致细胞凋亡。这表明AD中有氧糖酵解的增加可能导致该疾病中观察到的神经元功能障碍和毒性。我们推测，Ablation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。由于许多以前的研究没有区分神经元和神经胶质的贡献，也没有检查突触代谢变化的影响，我们正在使用允许精确定位测量的工具。在目标1中，我们将开发一种测定方法，使用基于FRET的葡萄糖传感器测量亚细胞分辨率的有氧糖酵解，并使用质谱法对表征良好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，神经元细胞体和胶质细胞中的葡萄糖水平。一旦我们优化了我们的测定，我们将测量Ablation对细胞体和突触神经元中葡萄糖代谢的影响。通过控制葡萄糖的摄取和消耗，我们将量化葡萄糖通过有氧糖酵解和其他代谢途径的流量，以确定Ablation如何改变代谢。在目标2中，我们将深入研究有氧糖酵解改变的生物能量效应，以确定代谢变化如何在功能上影响神经元。我们的实验室已经开发出一种检测突触ATP水平的方法。该测定使用基于FRET的ATP传感器区分线粒体和糖酵解衍生的ATP。我们将测试腺苷三磷酸对ATP水平的影响，并在功能上，其对突触传递与vGlut 1-pHluorin结构的影响。在目标3中，我们将在体内研究AD小鼠模型中有氧糖酵解是否发生变化，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光谱测试AD小鼠模型是否显示有氧糖酵解增加。然后，我们将通过敲除海马中的PKM 1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学评估来测试有氧糖酵解增加是否有害。在这个项目完成后，我们将确定是否Aesthetic增加有氧糖酵解，以及这如何有助于AD模型的毒性，测试潜在的治疗目标。我们还将开发出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.