Imaging cellular energy metabolism in vivo using fluorescent biosensors

使用荧光生物传感器对体内细胞能量代谢进行成像

• 批准号: 9115479
• 负责人: Carolina Lahmann
• 金额: $ 5.61万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115479
• 关键词: Accelerometer; Agonist; Apoptosis; Area; Astrocytes; BCL2 gene; Behavior; Biosensor; Blood; Brain; Carbohydrates; Cell Death; Cells; Cellular Metabolic Process; Cephalic; Cerebrospinal Fluid; Contralateral; Cultured Cells; Diet; Energy Metabolism; Epilepsy; Fatty acid glycerol esters; Fellowship; Fire - disasters; Fluorescence; Functional disorder; Genes; Glucose; Glycolysis; Goals; Image; Imaging Techniques; Implant; Individual; Ketone Bodies; Kinetics; Knock-out; Knockout Mice; Knowledge; Life; Link; Metabolic; Metabolic Pathway; Metabolism; Methods; Mitochondria; Modeling; Monitor; Mus; NADH; Neurologic; Neurons; Parkinson Disease; Pathway interactions; Physiological; Play; Point Mutation; Procedures; Proteins; Reading; Reporting; Research; Resistance; Resolution; Rest; Role; Sampling; Seizures; Slice; Somatosensory Cortex; Source; Time; Tissues; Translating; Vibrissae; Wild Type Mouse; Work; barrel cortex; brain cell; brain metabolism; cell growth regulation; cell type; cellular imaging; in vivo; insight; interest; live cell imaging; metabolic abnormality assessment; mouse model; neuronal excitability; optical imaging; preference; public health relevance; relating to nervous system; response; sensor; somatosensory; two-photon

 DESCRIPTION (provided by applicant): Metabolism provides the vital energy and building blocks necessary for the physiological function of all cells. The brain is one of the most metabolically demanding tissues, and cellular metabolic dysfunction is a hallmark of devastating neurological conditions ranging from epilepsy to Parkinson's Disease. Although the components of the core metabolic pathways have been known for decades, little is known about their moment-to-moment behavior and interactions in living cells. To better understand why metabolic dysfunction occurs in diseased states, it is crucial to first establish how energy metabolism of brain cells behaves in vivo. Traditional approaches to study cellular metabolism lack the temporal and spatial resolution required to examine real-time metabolism in native tissues. These barriers can be overcome by using genetically-encoded fluorescent biosensors. Recently created sensors that report the ATP/ADP ratio and the NADH/NAD+ ratio enable us to monitor energy metabolism in living brain cells. The cytosolic ATP/ADP ratio reflects the overall energetic state of the cell, while the cytosolic NADH/NAD+ ratio provides a read-out of glycolytic activity. Monitoring these two key metabolites in vivo will provide us unprecedented insight into the metabolic activities of brain cells during resting and active conditions in the intact brain. Te proposed research combines the use of fluorescent ATP and NADH biosensors with advanced optical imaging techniques to study the energy metabolism of neurons and astrocytes in vivo. The goal of Aim 1 is to characterize the dynamics of NADH and ATP levels in these cell types in vivo during resting and active states. This work will also examine whether glycolysis is activated in both cell types in response to physiological neuronal activity. The goal of Aim 2 is to determine whether NADH and ATP levels in neurons and astrocytes in vivo are altered in a mouse model where glucose is not the preferred mitochondrial fuel. This mouse model displays seizure resistance and characterizing its cellular neural metabolism may provide valuable insight into how metabolic modulation can regulate neuronal excitability.

 描述（由适用提供）：新陈代谢提供了所有细胞的身体功能所需的重要能量和构件。大脑是最需要代谢的组织之一，细胞代谢功能障碍是神经系统疾病的毁灭性疾病的标志，从癫痫到帕金森氏病。尽管核心代谢途径的成分数十年来一直闻名，但对它们的瞬间行为和活细胞中的相互作用知之甚少。为了更好地理解为什么代谢功能障碍发生在解散状态下，至关重要的是首先确定脑细胞的能量代谢如何在体内表现。研究细胞代谢的传统方法缺乏检查天然组织中实时代谢所需的临时和空间分辨率。可以使用遗传编码的荧光生物传感器来克服这些障碍。最近创建的传感器报告了ATP/ADP比和NADH/NAD+比率使我们能够监测活体细胞中的能量代谢。胞质ATP/ADP比反映了细胞的总能量状态，而胞质NADH/NAD+比提供了糖酵解活性的读出。监测体内这两个关键代谢产物将为我们对完整大脑的静止和活跃状态期间脑细胞代谢活性提供前所未有的见解。 TE提出的研究结合了荧光ATP和NADH生物传感器与晚期光学成像技术的使用，以研究体内神经元和星形胶质细胞的能量代谢。 AIM 1的目的是表征在静止和活跃状态期间这些细胞类型中NADH和ATP水平的动力学。这项工作还将检查是否响应物理神经元活性，在两种细胞类型中都激活了糖酵解。 AIM 2的目的是确定在体内神经元和星形胶质细胞中的NADH和ATP水平是否在小鼠模型中改变了葡萄糖不是首选的线粒体燃料。该小鼠模型表现出癫痫发作的抗性并表征其细胞神经元代谢，可能会为代谢调节如何调节神经元令人兴奋提供宝贵的见解。