A Cdc20-APC ubiquitin signaling pathway regulates presynaptic differentiation

The assembly of synapses is important for the establishment of neuronal circuitry in the brain. Presynaptic axonal differentiation is an essential step in synapse formation and remodeling. The assembly of presynaptic sites is temporally coordinated during development. However, the cell-intrinsic mechanisms that orchestrate the timing of presynaptic axonal differentiation are poorly understood.;The cerebellar cortex offers a robust system for the study of presynaptic development. Granule neurons project parallel fiber axons that synapse onto Purkinje neuron dendrites. Electron microscopy studies have revealed that parallel fibers enlarge to form "varicose" presynaptic specializations at sites of contact with Purkinje dendritic spines. In order to identify the mechanisms that regulate the development of presynaptic sites, I have developed assays to visualize presynaptic sites in primary cerebellar granule neurons and in vivo in the rat cerebellar cortex. Using these assays, I have found that both ubiquitin signaling and activity-dependent transcription play key roles in the control of presynaptic differentiation.;The first regulator of presynaptic differentiation I uncovered is the E3 ubiquitin ligase Cdc20-APC, which originally was identified for its critical role in progression through the cell cycle. I found that knockdown of Cdc20 and knockdown of the core APC subunit APC2 by RNA interference in postmitotic neurons impairs the assembly of presynaptic sites, suggesting that Cdc20-APC promotes presynaptic differentiation in neurons in the mammalian brain. Interestingly, I have also identified the transcription factor NeuroD2 as a neuron-specific substrate of Cdc20-APC. I found that NeuroD2 suppresses presynaptic differentiation during early development. However, with neuronal maturation, Cdc20-APC degrades NeuroD2 to trigger the formation of new presynaptic sites.;In addition to its regulation by the ubiquitin-proteasome system, NeuroD2-dependent transcription is also activated by calcium influx in response to neuronal activity. I found that calcium influx through L-type voltage gated calcium channels (VGCCs) suppresses presynaptic differentiation, phenocopying the function of NeuroD2. Importantly, a calcium responsive site of phosphorylation on NeuorD2 at Serine 315 is necessary for NeuroD2's ability to suppress presynaptic differentiation.;Together, these findings define a Cdc20-APC ubiquitin signaling pathway and an activity-dependent signaling pathway which converge upon NeuroD2 to orchestrate presynaptic development in the mammalian brain.