AbstractThe regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin dependent tagging and degradation through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated and whether SVs are cleared en-mass together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real time to support synaptic health. To address these questions, we have developed an imaging based system that simultaneously tags presynaptic proteins while monitoring autophagy. Moreover, by tagging SV proteins with a light activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control the damage to specific SV proteins and assess their consequence to autophagy mediated clearance mechanisms and synaptic function. Our results show that, in mouse hippocampal neurons, presynaptic autophagy can be induced in as little as 5-10 minutes and eliminates primarily the damaged protein rather than the SV en-mass. Importantly, we also find that autophagy is essential for synaptic function, as light-induced damage to e.g. Synaptophysin only compromises synaptic function when autophagy is simultaneously blocked. These data support the concept that presynaptic boutons have a robust highly regulated clearance system to maintain not only synapse integrity, but also synaptic function.Significance StatementThe real-time surveillance and clearance of synaptic proteins is thought to be vital to the health, functionality and integrity of vertebrate synapses and is compromised in neurodegenerative disorders, yet the fundamental mechanisms regulating these systems remain enigmatic. Our analysis reveals that presynaptic autophagy is a critical part of a real-time clearance system at glutamatergic synapses capable of responding to local damage of synaptic vesicle proteins within minutes and to be critical for the ongoing functionality of these synapses. These data indicate that synapse autophagy is not only locally regulated but also crucial for the health and functionality of vertebrate presynaptic boutons.
Light induced synaptic vesicle autophagy