Association of Nwk with SNX16 promotes down-regulation of synaptic growth signaling at the interface between early and recycling endosomes.

Summary Synaptic plasticity is a fundamental feature of the nervous system that allows adaptation to changing behavioral environments. Most studies of synaptic plasticity have examined the regulated trafficking of postsynaptic glutamate receptors that generates alterations in synaptic transmission. Whether and how changes in the presynaptic release machinery contribute to neuronal plasticity is less clear. The SNARE complex mediates neurotransmitter release in response to presynaptic Ca++ entry. Here we show that the SNARE fusion clamp Complexin undergoes activity-dependent phosphorylation that alters the basic properties of neurotransmission in Drosophila. Retrograde signaling following stimulation activates PKA-dependent phosphorylation of the Complexin C-terminus that selectively and transiently enhances spontaneous release. Enhanced spontaneous release is required for activity-dependent synaptic growth. These data indicate that SNARE-dependent fusion mechanisms can be regulated in an activity-dependent manner and highlight the key role of spontaneous neurotransmitter release as a mediator of functional and structural plasticity.

Complexins are small α-helical proteins that modulate neurotransmitter release by binding to SNARE complexes during synaptic vesicle exocytosis. They have been found to function as fusion clamps to inhibit spontaneous synaptic vesicle fusion in the absence of Ca2+, while also promoting evoked neurotransmitter release following an action potential. Complexins consist of an N-terminal domain and accessory α-helix that regulate the activating and inhibitory properties of the protein, respectively, and a central α-helix that binds the SNARE complex and is essential for both functions. In addition, Complexins contain a largely unstructured C-terminal domain whose role in synaptic vesicle cycling is poorly defined. Here, we demonstrate that the C-terminus of Drosophila Complexin (DmCpx) regulates localization to synapses and that alternative splicing of the C-terminus can differentially regulate spontaneous and evoked neurotransmitter release. Characterization of the single DmCpx gene by mRNA analysis revealed expression of two alternatively expressed isoforms, DmCpx7A and DmCpx7B, which encode proteins with different C-termini that contain or lack a membrane tethering prenylation domain. The predominant isoform, DmCpx7A, is further modified by RNA editing within this C-terminal region. Functional analysis of the splice isoforms showed that both are similarly localized to synaptic boutons at larval neuromuscular junctions, but have differential effects on the regulation of evoked and spontaneous fusion. These data indicate that the C-terminus of Drosophila Complexin regulates both spontaneous and evoked release though separate mechanisms and that alternative splicing generates isoforms with distinct effects on the two major modes of synaptic vesicle fusion at synapses.

Genetic studies in Drosophila have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in radish (rsh) mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways leading to these forms of memory may share the cAMP cascade critical for associative learning. Dunce, which encodes a cAMP-specific phosphodiesterase, and rutabaga, which encodes an adenylyl cyclase, both disrupt short-term memory. Amnesiac encodes a pituitary adenylyl cyclase-activating peptide homolog and is required for middle-term memory. Here, we demonstrate that the Radish protein localizes to the cytoplasm and nucleus and is a PKA phosphorylation target in vitro. To characterize how these plasticity pathways may manifest at the synaptic level, we assayed synaptic connectivity and performed an expression analysis to detect altered transcriptional networks in rutabaga, dunce, amnesiac, and radish mutants. All four mutants disrupt specific aspects of synaptic connectivity at larval neuromuscular junctions (NMJs). Genome-wide DNA microarray analysis revealed ∼375 transcripts that are altered in these mutants, suggesting defects in multiple neuronal signaling pathways. In particular, the transcriptional target Lapsyn, which encodes a leucine-rich repeat cell adhesion protein, localizes to synapses and regulates synaptic growth. This analysis provides insights into the Radish-dependent ARM pathway and novel transcriptional targets that may contribute to memory processing in Drosophila.