Metabolic coupling of neuronal ion transport

神经元离子转运的代谢耦合

• 批准号: 10155101
• 负责人: Dylan John Meyer
• 金额: $ 6.6万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10155101
• 关键词: Active Biological Transport; Active Ion Transport; Acute; Address; Affect; Bioinformatics; Biosensor; Brain; Buffers; CKB gene; CRISPR/Cas technology; Carbohydrates; Carrier Proteins; Cell membrane; Cells; Consumption; Coupled; Couples; Coupling; Creatine Kinase; Cytoplasmic Granules; Data; Data Analyses; Dependence; Development; Dyes; Electrophysiology (science); Environment; Enzymes; Epilepsy; Event; Feedback; Fellowship; Functional disorder; Genes; Glycolysis; High Fat Diet; Hippocampus (Brain); Homeostasis; Immersion; Individual; Ion Channel; Ion Transport; Ions; Isoenzymes; Journals; Knock-out; Knockout Mice; Knowledge; Leadership; Learning; Measures; Mediating; Membrane; Metabolic; Metabolic Pathway; Microscopy; Mitochondria; Na(+)-K(+)-Exchanging ATPase; Neurodegenerative Disorders; Neurons; Oxidative Phosphorylation; Pathology; Pharmacology; Process; Production; Pump; RNA; Reaction; Reducing diet; Research; Rest; Role; Signal Transduction; Slice; Source; Synapses; Therapeutic; Time; Tissues; Training; Transport Process; Work; Writing; adenylate kinase; brain cell; career; experience; experimental study; fluorescence lifetime imaging; inhibitor/antagonist; innovation; ion dynamics; ketogenic diet; medical schools; mitochondrial metabolism; neural circuit; neuronal excitability; neuronal metabolism; neurotransmission; postsynaptic; receptor; response; sensor; skills; two-photon; voltage gated channel

Proper energy utilization and management by the brain is essential for neurons to process information and communicate effectively. It is well known that active ion transport activities consume enormous quantities of ATP during neuronal signaling to restore plasma membrane ion gradients and maintain cellular excitability, but how active ion transport is fueled by specific metabolic pathways and ATP buffering mechanisms is still controversial. This research fellowship aims to study two aspects of energy management during neuronal signaling: 1) whether active ion transport preferentially couples to ATP produced from glycolysis or from oxidative phosphorylation, and 2) whether creatine kinase and adenylate kinase buffer ATP during periods of intense energy demand. The proposed experiments will utilize two-photon fluorescence lifetime imaging of genetically encoded fluorescent biosensors and dyes to accurately quantify real-time metabolite and ion dynamics in hippocampal dentate granule neurons of acute brain slices following synaptic stimulation. Pharmacological strategies will then be employed to tease apart the contributions of specific active transport activities, ion channels, and metabolic pathways to the metabolite and ion sensor lifetime signals. Also, CRISPR-Cas9:sg RNA gene editing will be used to determine how ATP buffering in dentate granule neurons is affected by knockout of creatine kinase or adenylate kinase isozymes. Knowing whether neuronal active ion transport during signaling is differentially regulated by glycolysis or oxidative phosphorylation, and whether it is supported by ATP-buffering enzymes, is important for detailing how neuronal excitability is regulated by different metabolic fuels, and will have implications for understanding how the metabolic alterations resulting from a ketogenic diet – a very-low- carbohydrate, high-fat diet – are therapeutic for epilepsy. This knowledge will also help to determine the underlying pathophysiological mechanisms of neurodegenerative disorders that are associated with energetic dysfunction. This fellowship training plan contains numerous outside-the-lab activities to aid the awardee's scientific development and allow for continued learning, including courses on neural circuits, microscopy, bioinformatics skills for data analysis, scientific writing, lab leadership, and many others. Weekly department seminars and journal clubs will allow the awardee to learn about related research and receive feedback about their data and hypotheses. The research environment of Harvard Medical School will provide the awardee with an immersive learning and training experience that will facilitate their transition into the next stage of their career.

大脑的适当能量利用和管理对于神经元进行处理和有效沟通至关重要。众所周知，主动离子转运活性在神经元信号传导过程中消耗大量的ATP以恢复质膜离子梯度并保持细胞刺激性，但是仍有争议的特定代谢途径和ATP缓冲机制为主动离子传输如何促进。这项研究奖学金旨在研究神经元信号传导期间能量管理的两个方面：1）活性离子转运是否优先伴侣与糖酵解或氧化物磷酸化产生的ATP以及2）在强烈的能量需求期间是否会产生激酶和腺苷酸激酶缓冲液ATP。提出的实验将利用遗传编码的荧光生物传感器和染料的两光子荧光寿命成像，以准确量化突触刺激后急性脑切片的海马牙齿颗粒神经元中的实时代谢物和离子动力学。然后，将采用药理学策略来教授特定主动运输活动，离子渠道以及代谢物和离子传感器寿命信号的代谢途径的贡献。同样，将使用CRISPR-CAS9：SG RNA基因编辑来确定齿状颗粒神经元中的ATP缓冲如何受到创造激酶或腺苷酸激酶同工酶的敲除。知道信号过程中神经元活性离子传输是否受糖酵解或氧化磷酸化的不同调节，以及是否得到ATP缓冲酶的支持，对详细介绍神经元令人兴奋的神经元如何受到不同代谢燃料的调节非常重要癫痫。这些知识还将有助于确定与能量功能障碍相关的神经退行性疾病的潜在病理生理机制。该奖学金培训计划包含了许多外部活动，以帮助获奖者的科学发展，并允许继续学习，包括有关神经循环的课程，显微镜，显微镜，生物信息学技能，用于数据分析，科学写作，实验室领导力等。每周的部门半少数和期刊俱乐部将允许获奖者了解相关研究，并收到有关其数据和假设的反馈。哈佛医学院的研究环境将为获奖者提供身临其境的学习和培训经验，这将有助于他们过渡到其职业生涯的下一阶段。