Synaptophysin sustains presynaptic performance by preserving vesicular synaptobrevin‐II levels

The two most abundant molecules on synaptic vesicles (SVs) are synaptophysin and synaptobrevin‐II (sybII). SybII is essential for SV fusion, whereas synaptophysin is proposed to control the trafficking of sybII after SV fusion and its retrieval during endocytosis. Despite controlling key aspects of sybII packaging into SVs, the absence of synaptophysin results in negligible effects on neurotransmission. We hypothesised that this apparent absence of effect may be because of the abundance of sybII on SVs, with the impact of inefficient sybII retrieval only revealed during periods of repeated SV turnover. To test this hypothesis, we subjected primary cultures of synaptophysin knockout neurons to repeated trains of neuronal activity, while monitoring SV fusion events and levels of vesicular sybII. We identified a significant decrease in both the number of SV fusion events (monitored using the genetically encoded reporter vesicular glutamate transporter‐pHluorin) and vesicular sybII levels (via both immunofluorescence and Western blotting) using this protocol. This revealed that synaptophysin is essential to sustain both parameters during periods of repetitive SV turnover. This was confirmed by the rescue of presynaptic performance by the expression of exogenous synaptophysin. Importantly, the expression of exogenous sybII also fully restored SV fusion events in synaptophysin knockout neurons. The ability of additional copies of sybII to fully rescue presynaptic performance in these knockout neurons suggests that the principal role of synaptophysin is to mediate the efficient retrieval of sybII to sustain neurotransmitter release. Despite controlling the packaging of the essential fusogenic protein synaptobrevin II onto synaptic vesicles, the absence of synaptophysin results in surprisingly mild effects on neurotransmission. We hypothesised that the abundance of vesicular synaptobrevin may mean that effects on exocytosis only appear during periods of heightened activity. To test this, we subjected synaptophysin knockout neurons to repeated trains of activity, and revealed that synaptophysin is essential to sustain the number of fusion events and vesicular synaptobrevin levels during periods of repetitive vesicle turnover. Therefore, the principal role of synaptophysin is to mediate the efficient retrieval of synaptobrevin to sustain neurotransmitter release.