Metabolic coupling of neuronal ion transport

大脑适当的能量利用和管理是神经元处理信息和有效沟通的必要条件。众所周知，在神经元信号传递过程中，活性离子转运活动消耗大量的ATP来恢复质膜离子梯度和维持细胞的兴奋性，但活性离子转运如何通过特定的代谢途径和ATP缓冲机制来促进仍然存在争议。本研究旨在研究神经元信号传导过程中能量管理的两个方面：1)活性离子转运是否优先与糖酵解或氧化磷酸化产生的ATP偶联；2)肌酸激酶和腺苷酸激酶是否在能量需求旺盛时缓冲ATP。该实验将利用基因编码荧光生物传感器和染料的双光子荧光寿命成像，准确定量突触刺激后急性脑切片海马齿状颗粒神经元的实时代谢物和离子动力学。然后将采用药理学策略来梳理特定主动转运活动、离子通道和代谢途径对代谢物和离子传感器寿命信号的贡献。此外，CRISPR-Cas9:sg RNA基因编辑将用于确定敲除肌酸激酶或腺苷酸激酶同工酶如何影响齿状颗粒神经元中的ATP缓冲。了解信号传导过程中神经元活性离子运输是否受到糖酵解或氧化磷酸化的不同调节，以及它是否受到atp缓冲酶的支持，对于详细说明神经元兴奋性如何受到不同代谢燃料的调节非常重要，并将对理解生酮饮食（极低碳水化合物，高脂肪饮食）导致的代谢改变如何治疗癫痫具有重要意义。这一知识也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许多实验室外的活动，以帮助获奖者的科学发展，并允许继续学习，包括神经回路，显微镜，生物信息学技能的数据分析，科学写作，实验室领导等课程。每周的部门研讨会和期刊俱乐部将使获奖者了解相关研究，并收到有关他们的数据和假设的反馈。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体验，这将有助于他们过渡到职业生涯的下一个阶段。

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.