The 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 masse 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 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 of either sex, presynaptic autophagy can be induced in as little as 5-10 min and eliminates primarily the damaged protein rather than the SV en masse. Importantly, we also find that autophagy is essential for synaptic function, as light-activated damage to, for example, 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.

Mechanisms regulating the turnover of synaptic vesicle (SV) proteins are not well understood. They are thought to require poly-ubiquitination and degradation through proteasome, endo-lysosomal or autophagy-related pathways. Bassoon was shown to negatively regulate presynaptic autophagy in part by scaffolding Atg5. Here, we show that increased autophagy in Bassoon knockout neurons depends on poly-ubiquitination and that the loss of Bassoon leads to elevated levels of ubiquitinated synaptic proteins per se. Our data show that Bassoon knockout neurons have a smaller SV pool size and a higher turnover rate as indicated by a younger pool of SV2. The E3 ligase Parkin is required for increased autophagy in Bassoon-deficient neurons as the knockdown of Parkin normalized autophagy and SV protein levels and rescued impaired SV recycling. These data indicate that Bassoon is a key regulator of SV proteostasis and that Parkin is a key E3 ligase in the autophagy-mediated clearance of SV proteins.

Loss of function of the active zone protein Piccolo has recently been linked to a disease, Pontocerebellar Hypoplasia type 3, which causes brain atrophy. Here, we address how Piccolo inactivation in rat neurons adversely affects synaptic function and thus may contribute to neuronal loss. Our analysis shows that Piccolo is critical for the recycling and maintenance of synaptic vesicles. We find that boutons lacking Piccolo have deficits in the Rab5/EEA1 dependent formation of early endosomes and thus the recycling of SVs. Mechanistically, impaired Rab5 function was caused by reduced synaptic recruitment of Pra1, known to interact selectively with the zinc finger domains of Piccolo. Importantly, over-expression of GTPase deficient Rab5 or the Znf1 domain of Piccolo restores the size and recycling of SV pools. These data provide a molecular link between the active zone and endosome sorting at synapses providing hints to how Piccolo contributes to developmental and psychiatric disorders.

Piccolo, a presynaptic active zone protein, is best known for its role in the regulated assembly and function of vertebrate synapses. Genetic studies suggest a further link to several psychiatric disorders as well as Pontocerebellar Hypoplasia type 3 (PCH3). We have characterized recently generated Piccolo KO (Pclogt/gt) rats. Analysis of rats of both sexes revealed a dramatic reduction in brain size compared with WT (Pclowt/wt) animals, attributed to a decrease in the size of the cerebral cortical, cerebellar, and pontine regions. Analysis of the cerebellum and brainstem revealed a reduced granule cell layer and a reduction in size of pontine nuclei. Moreover, the maturation of mossy fiber afferents from pontine neurons and the expression of the α6 GABAAreceptor subunit at the mossy fiber-granule cell synapse are perturbed, as well as the innervation of Purkinje cells by cerebellar climbing fibers. Ultrastructural and functional studies revealed a reduced size of mossy fiber boutons, with fewer synaptic vesicles and altered synaptic transmission. These data imply that Piccolo is required for the normal development, maturation, and function of neuronal networks formed between the brainstem and cerebellum. Consistently, behavioral studies demonstrated that adultPclogt/gtrats display impaired motor coordination, despite adequate performance in tasks that reflect muscle strength and locomotion. Together, these data suggest that loss of Piccolo function in patients with PCH3 could be involved in many of the observed anatomical and behavioral symptoms, and that the further analysis of these animals could provide fundamental mechanistic insights into this devastating disorder.SIGNIFICANCE STATEMENTPontocerebellar Hypoplasia Type 3 is a devastating developmental disorder associated with severe developmental delay, progressive microcephaly with brachycephaly, optic atrophy, seizures, and hypertonia with hyperreflexia. Recent genetic studies have identified non-sense mutations in the coding region of the PCLO gene, suggesting a functional link between this disorder and the presynaptic active zone. Our analysis of Piccolo KO rats supports this hypothesis, formally demonstrating that anatomical and behavioral phenotypes seen in patients with Pontocerebellar Hypoplasia Type 3 are also exhibited by these Piccolo deficient animals.