ER-associated control of axonal vesicle trafficking during neuronal development

Axonal vesicle trafficking is essential for presynaptic assembly, yet the intracellular structures that spatially and temporally regulate vesicle transport during neuronal development remain unclear. Here we identify previously uncharacterized membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and both synaptic vesicle precursors (SVPs) and dense-core vesicles (DCVs). Using super-resolution and cryo-electron microscopy, we reveal that vesicle transport undergoes a transient developmental slowdown in the axons of rat hippocampal neurons associated with increased vesicle tethering to the ER. Using proximity biotinylation, we identify the ER protein PRG1 as a developmentally regulated factor enriched at these sites that modulates vesicle mobility. Notably, a disease-associated PRG1 mutant fails to regulate vesicle trafficking. Finally, we demonstrate that PRG1 modulation of vesicle trafficking serves as a developmental checkpoint by delaying synapse formation and preventing premature network activity. Together, our findings establish ER-vesicle contact sites as a previously unrecognized layer of control of axonal trafficking and link intracellular organization to the timing of synapse assembly.