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.

Synaptic vesicle (SV) release probability (Pr), determines the steady state and plastic control of neurotransmitter release. However, how diversity in SV composition arises and regulates the Pr of individual SVs is not understood. We found that modulation of the copy number of the noncanonical vesicular SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor), vesicle-associated membrane protein 4 (VAMP4), on SVs is key for regulating Pr. Mechanistically, this is underpinned by its reduced ability to form an efficient SNARE complex with canonical plasma membrane SNAREs. VAMP4 has unusually high synaptic turnover and is selectively sorted to endolysosomes during activity-dependent bulk endocytosis. Disruption of endolysosomal trafficking and function markedly increased the abundance of VAMP4 in the SV pool and inhibited SV fusion. Together, our results unravel a new mechanism for generating SV heterogeneity and control of Pr through coupling of SV recycling to a major clearing system that regulates protein homeostasis.