The spatiotemporal regulation of neuronal metabolism is a critical determinant of proper brain function. Investigating the underlying mechanisms governing the dynamic interplay between neuronal metabolic processes, including glycosome formation on mitochondria through O-GlcNAcylation, and their spatial distribution and temporal coordination is therefore of utmost importance. I utilized a combination of primary cell lines and mouse models, computational modeling, and advanced imaging techniques to elucidate the critical components and pathways involved in the regulation of energy supply, demand, and signaling in neurons. My findings highlight the importance of metabolic coupling between glycolysis and oxidative phosphorylation, as well as the role of post-translational modifications (PTMs) such as O-GlcNAcylation in sensing and responding to changes in glucose availability. Specifically, I demonstrate the critical role of glycosome formation on mitochondria through O-GlcNAcylation in spatiotemporal regulation of neuronal metabolism. By providing a comprehensive understanding of the spatiotemporal regulation of neuronal metabolism, this study offers insights into potential therapeutic targets for brain disorders linked to metabolic dysregulation. Ultimately, my work contributes to advancing the field's understanding of the intricate mechanisms underlying neuronal metabolism and its implications for brain function and health.

Glucose metabolism starts with the activity of the first rate-limiting step enzyme Hexokinase (HK). In the brain, which the major energy source is glucose, HK-1 is the dominant isoform, and mostly associated with the mitochondrial outer membrane. This positioning of HK1 is critical, because it couples glucose metabolism to the energy production pathways of mitochondria. Here, we report a new molecular mechanism that regulates the mitochondrial positioning and activity of HK-1 via the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes a reversible posttranslational modification of proteins by adding a GlcNAc sugar moiety to serine and threonine residues. The catalytic activity of OGT is regulated by intracellular UDP-GlcNAc concentrations, which fluctuate proportionally in response to nutrient flux through the hexosamine pathway. In this study, We show that HK-1 is dynamically modified with O-GlcNAc at its regulatory domain. O-GlcNAcylation of HK-1 is elevated when cells were transfected with OGT or treated with TMG. We further characterized O-GlcNAc modification of HK1 recruited it on mitochondria and increases its enzymatic activity. Our findings may reveal key molecular pathways that couple neuronal metabolism to mitochondrial function via OGT, and how their dysregulation leads to neurological disorders.