UC San Diego

Maintaining homeostasis through energy metabolism is a crucial function all cells must perform. This is especially true in the context of the brain, where even acute perturbations in energy levels can result in permanent brain damage. Discussed here are two separate projects focused on improving the understanding of and ability to examine neuronal energy metabolism. The first chapter discuss the localization of the rate-limiting enzyme of glycolysis, phosphofructokinase-1, in mammalian neurons. Evidence is shown of enrichment between phosphopfructokinase-1 and mitochondria, suggesting a potential spatial coupling between the processes of glycolysis and oxidative phosphorylation. The second project discusses the development of a program to efficiently normalize metabolic flux data through cell counts without a dedicated cell counting instrument. Metabolic flux assays are a powerful tool to gain insight into the rates of glycolysis and oxidative phosphorylation change in response to different experimental conditions. However, variance in the plated cell density of each well demands normalization to obtain accurate results. The software developed, SHCellAnalyzer, is comprised of two individual programs, an imageJ macro which physically counts the cells and a python script which takes those cell counts and extrapolates them to represent the entire well. Together these programs efficiently and accurately produce cell count estimates which can consistently be used to normalize metabolic flux data.