Products of the Drosophila stoned gene regulate synaptic vesicle cycling and synaptic plasticity

Abstract

Chemical synaptic transmission requires the regulated release of neurotransmitter from synaptic vesicles stored within the presynaptic terminal. Synaptic vesicles release their contents by exocytosis, and are subsequently recycled by endocytosis and reassembly of synaptic vesicle components deposited in the plasma membrane. Previous studies have suggested that products of the Drosophila stoned gene regulate this cycling of synaptic vesicles (Petrovich et al., 1993; Andrews et al., 1996). Stoned encodes two novel proteins, stonedA and stonedB, which possess sequence motifs shared by proteins involved in intracellular vesicle traffic. Via analyses of Drosophila larval neuromuscular synapses, the work presented here provides the first direct evidence that stoned proteins regulate synaptic vesicle cycling. First, stonedA and stonedB are enriched at presynaptic terminals, and mutations of stoned decrease presynaptic levels of the stoned proteins. Second, all stoned mutations examined here disrupt neurotransmitter release, and cause mislocalization of synaptotagmin to the plasma membrane. Though this mislocalization suggests defective retrieval of synaptic vesicle components from the plasma membrane, the viable EMS-induced stnᵗˢ² and stnᶜ mutations do not decrease the supply of synaptic vesicles or alter the assembly of morphologically normal vesicles. Thus, impaired neurotransmitter release at stnᵗˢ² and stnᶜ synapses either arises from subtle defects of synaptic vesicle recycling, or indicates a role for the stoned proteins in synaptic vesicle exocytosis. The stn⁸ᴾ¹ mutation severely reduces synaptic transmission, decreases synaptic vesicle density and increases synaptic vesicle size. Thus, altered neurotransmitter release at stn⁸ᴾ¹ synapses certainly arises in part from defects in synaptic vesicle endocytosis. Unexpectedly, all three stoned mutants show overgrowth of the presynaptic terminal. In the stn⁸ᴾ¹ mutant, terminals have a large number of bud-like satellite boutons, also observed in Drosophila overexpressing the Amyloid Precursor Protein, a cell surface protein implicated in human Alzheimer's Disease. Thus, altered presynaptic structure in stoned mutants might indicate impaired endocytosis of cell-cell signaling molecules that regulate synapse growth. Overall, these data establish that stoned proteins regulate the trafficking of synaptic vesicles, and suggest that stoned proteins additionally regulate cellular trafficking pathways that influence synaptic plasticity.