Autophagy and its role in synapse function

The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin- dependent tagging and degradation of SV proteins 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, I have developed an imaging based system that simultaneously labels presynaptic proteins while monitoring the appearance of autophagosomes and/or late endosomes within the presynaptic bouton. Moreover, by tagging three synaptic proteins (Synaptophysin, Synaptotagmin, Synapsin) with a light-activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control the damage of specific SV proteins and assess its consequence to autophagy mediated clearance mechanisms and synaptic function. My results show that, in mouse hippocampal neurons, presynaptic autophagy can be induced in as little as 5-10 minutes (after Synaptophysin-Supernova bleaching) and eliminates primarily the damaged protein rather than the SV en-mass. Additionally, I could show that induction of ROS-activated presynaptic autophagy requires the close association of Supernova with SVs. Importantly, I also found that autophagy is essential for synaptic function, as ROS damage to e.g. Synaptophysin only compromises synaptic function when autophagy is simulaneously blocked, as measured in FM dye uptake and electrophysiology experiments. These data support the concept that presynaptic boutons have a robust, highly regulated clearance system to react in real-time to maintain not only synapse integrity, but also synapse function. Additionally, I was able to further characterize neurons from Bassoon KO mice, which depict higher autophagy levels at boutons and along axons compared to neurons from WT mice. Therefore, they may be a useful tool to study presynaptic autophagy. In my study, imaging based experiments confirmed that the autophagy-related phenotype in Bassoon KO neurons does not show a neurodegenerative progression and that the increase in autophagy is dependent on poly- ubiquitination as well as the autophagy-related protein Atg5. To summarize, I was able to provide new insights into the timing, the cargo and the function of presynaptic autophagy. Furthermore, my findings support the concept that functional autophagy is important for the clearance of damaged proteins, whereas excessively induced autophagy can be detrimental for synapse health. Thus, synaptic autophagy needs to be in balance