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.
Proper neuron function requires that asymmetric concentrations of ions be maintained across the plasma membrane, but the ways that neurons produce the enormous amount of energy that is needed for this task are still controversial. This project aims to determine where, within the neuron, the energy for maintaining ion homeostasis comes from by studying both the dependence of active ion transport proteins on ATP produced from glycolysis and from mitochondrial metabolism, and the role of ATP- buffering enzymes in sustaining adequate energy availability. The results of this work will provide fundamental information about the coupling between neuronal metabolism and ion homeostasis, and will have important implications for understanding both the pathophysiological mechanisms of neurodegenerative disorders associated with energy dysfunction and how the metabolic alterations resulting from a ketogenic diet ? a low-carbohydrate, high-fat diet ? are therapeutic for epilepsy.
