Synapse Formation

To identify neuronal pathways that control synapse formation and synaptic growth, we have conducted forward genetic screens for temperature-sensitive mutants that disrupt the process. Several Ca2+-dependent presynaptic proteins have been implicated in synaptic growth, but the source of Ca2+ and its regulation are unknown. In addition to retrograde signaling, we have found that presynaptic Ca2+ influx through N-type channels participates in synaptic growth via signaling pathways that are distinct from those that mediate neurotransmitter release. The opening of presynaptic N-type channels during robust synaptic activity allows presynaptic Ca2+ influx to modulate sprouting mechanisms that locally control synaptic remodeling. Linking presynaptic voltage-gated Ca2+ entry to downstream Ca2+-sensitive synaptic growth regulators provides an efficient activity-dependent mechanism for modifying synaptic strength.

Several cell adhesion proteins and synaptic growth regulators reside adjacent to the active zone where presynaptic Ca2+ channels localize, in domains termed peri-active zones. We identified and characterized a peri-active zone signaling complex that controls synapse morphology and axonal branching through regulation of actin dynamics and endosomal trafficking of growth factors by the recycling endosome F-BAR/SH3 protein Nervous Wreck (NWK). The work on NWK was one of the first characterizations of a member of the now widely studied F-BAR protein family, a group of cytosolic proteins that bend membranes. NWK interacts with SNX16, which resides on early signaling endosomes and promotes synaptic growth through the Wingless and BMP pathways. These experiments defined a presynaptic trafficking pathway mediated by Snx16, NWK and the ESCRT complex that regulates synaptic growth signaling by modulating membrane dynamics and signal output at the interface between endosomal compartments.

Recent findings from our lab and others have begun to identify the machinery that functions to sense Ca2+ rises in nerve terminals during activity and trigger information transfer between neurons through vesicle fusion and the release of neurotransmitters. Presynaptically, Ca2+ influx following stimulation drives a synchronous phase of SV fusion that occurs within milliseconds and a slower asynchronous component that can last for hundreds of milliseconds depending on the synapse and firing pattern of the neuron. Current models suggest Ca2+ influx drives SV fusion by binding to the conserved Synaptotagmin (Syt) family of vesicular Ca2+ sensors. Syt1 has been shown to function as the synchronous sensor, while its homolog Syt7 may act as the asynchronous sensor, although this model is still controversial. Postsynaptically, Ca2+ influx through neurotransmitter receptors or voltage-gated channels triggers synapse-specific plasticity. We recently discovered that Synaptotagmin 4 (Syt4) is an evolutionarily conserved Syt homolog that localizes to the postsynaptic compartment and regulates Ca2+-dependent postsynaptic vesicle trafficking and the release of retrograde signals that trigger synaptic growth and plasticity. In contrast to SV release, we know little about the regulation of postsynaptic vesicle fusion and how retrograde signals modulate synapse biology. By defining the molecular machinery for postsynaptic release, we hope to generate new models to advance the field of retrograde signaling into a more mechanistic analysis of its function. Using newly developed tools to visualize presynaptic and postsynaptic vesicle fusion, we can take advantage of genetic approaches in Drosophila to dissect synaptic signaling at a level of resolution few other model systems can offer.