Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins. Synopsis Live‐cell immunolabeling in combination with metabolic imaging reveals that ageing synaptic vesicles are inactivated after approximately 200 rounds of release in primary hippocampal cultures. Ageing synaptic vesicles are inactivated after ˜ 200 rounds of release. Inactivation is followed by a phase of inactivity before degradation. Inactivation likely precedes accumulation of damage. Inactivation may be caused by contamination of vesicles with SNAP25. SNAP25 may be involved in triggering degradation via fusion to endosomes. Graphical Abstract Live‐cell immunolabeling and metabolic imaging of cultured neurons shows that synaptic vesicles remain in use for about 24 h and become fusion‐incompetent only after some 200 rounds of exo‐ and endocytosis.