Hexokinase-1 Activity Modulates Synaptic Vesicle Docking but not Endocytic Recycling

Synaptic vesicle (SV) recycling is fundamental to neurotransmission and is highly energy-dependent. Glycolysis, initiated by hexokinase (HK), is a key source of ATP at synapses. Previous work using pHluorin assays showed that inhibiting hexokinase reduces SV numbers, but the ultrastructural basis for this defect remained unclear. We hypothesized that HK-1 knockdown would impair either SV exocytosis, leading to an accumulation of docked vesicles, or endocytic recycling, leading to SV depletion. Using high-pressure freezing and electron microscopy, we analyzed the ultrastructure of hippocampal synapses expressing either wild-type (WT) HK-1 or an enzymatically dead mutant (T259A). We found no significant change in the total number of cytosolic, tethered, or large endocytic vesicles, indicating that endocytic recycling is not impaired. However, we observed a significant increase in the number of docked vesicles in HK-1(T259A) synapses. These results suggest that HK-1 activity and glycolytic ATP production are specifically required for the vesicle fusion step of exocytosis, rather than for endocytic retrieval, revealing a critical energy checkpoint in the synaptic vesicle cycle.