Neurons communicate with their target cells primarily through the regulated exocytosis of chemical neurotransmitters. While the cellular physiology of neurotransmitter release is well understood from classical work carried out at the neuromuscular junction, until relatively recently little was known of the molecular components underlying this process, particularly those promoting the targeting and regulated fusion of synaptic vesicles with the nerve terminal plasma membrane. In the early 1990's studies of regulated and constitutive secretory mechanisms led to the identification of candidate polypeptides that may function in this process, and a model, known as the SNARE hypothesis, proposing functions for these polypeptides. According to this model, synaptic vesicle targeting to and docking at release sites involves the assembly of a 7S protein complex consisting of vesicle- and target-membrane proteins, known collectively as SNAREs. This 7S SNARE complex recruits the cytosolic SNAP (Soluble NSF Attachment Proteins) and NSF (N-ethylmaleimide Sensitive Fusion protein) to form a 20S fusion complex. NSF then couples ATP hydrolysis to disassembly of the SNARE complex in a step postulated to initiate membrane fusion.

To explore fundamental tenets of the SNARE hypothesis model, we initiated studies on a Drosophila SNAP homolog and a pair of closely related Drosophila NSF homologs. Our work on one of the NSF homologs (NSF1) established that this gene was allelic with a pre-existing mutation conferring temperature-sensitive paralysis known as comatose. Previous work had shown that comatose mutants have a temperature-dependent block in synaptic transmission. Thus, our study provided the first definitive in vivo evidence that NSF functions in synaptic transmission. Subsequent studies of comatose mutants have revealed an activity-dependent defect in neurotransmitter release and an accumulation of docked synaptic vesicles at restrictive temperature, implying that NSF1 functions in a vesicle priming step downstream of synaptic vesicle docking. To explore the biochemical function of NSF we analyzed the abundance and subcellular distribution of SNARE complexes in comatose mutants. Results of this analysis demonstrated that SNARE complexes accumulate in the plasma membrane at restrictive temperature. Further, we have found that SNARE complexes persist in Drosophila mutants devoid of synaptic vesicles, indicating that SNARE complexes are not associated with docked synaptic vesicles, but rather reside free in the plasma membrane. Results from these studies indicate that NSF1 functions at the plasma membrane to disassemble a SNARE complex that forms prior to or during synaptic vesicle fusion. Further, our finding that SNARE complexes are only present in plasma membrane fractions (and not in synaptic vesicles) established that SNARE complex disassembly must precede recycling of the vesicle SNARE synaptobrevin into synaptic vesicles.

More recently we have generated mutations in a second Drosophila NSF gene (NSF2). In contrast to NSF1, which appears to be required only in the nervous system, NSF2 is primarily required in muscle. Electrophysiological analysis of NSF2 mutants reveals a severe reduction in both evoked release of neurotransmitter and in the amplitude of miniature excitatory postsynaptic currents. These results suggest that NSF2 functions in a postsynaptic capacity to regulate synaptic function. Further studies indicate that the postsynaptic defects in NSF2 mutants do not derive from altered postsynaptic receptor localization or abundance. However, structural analysis of synaptic terminals in NSF2 mutants has revealed a significant reduction in the number of large synaptic boutons (sites of neurotransmitter release) and possibly synaptic misrouting defects, indicating that NSF2 regulates the secretion of postsynaptic factors involved in the proper formation of synapses. We are currently investigating the possible involvement of candidate factors responsible for this phenotype.

We have also used mutational and transgenic approaches in Drosophila to investigate the effect of altered SNAP dosage on neurotransmitter release and SNARE complex metabolism. Our results indicate that reduced SNAP activity results in diminished neurotransmitter release and accumulation of a neural SNARE complex. Increased SNAP dosage results in defective synapse formation and a variety of tissue morphological defects without detectably altering the abundance of neural SNARE complexes or the efficiency of neurotransmitter release. The SNAP overexpression phenotypes are enhanced by mutations in other secretory components, and suppressed by co-overexpression of NSF1 or NSF2, indicating that these phenotypes derive from a specific perturbation of the secretory pathway. Our results indicate that SNAP promotes neurotransmitter release and SNARE complex disassembly but inhibits secretion when present at high abundance relative to NSF. We are currently testing models to explain these findings.

Finally, we have used classical genetic methods to identify novel components of the neurotransmitter release apparatus. One approach involves a screen for phototactic mutants using a genetic method allowing efficient production of mosaic flies homozygous for a mutagenized chromosome arm in the eye, but heterozygous elsewhere. Mutants recovered from this screen can be easily categorized using simple electroretinogram recording approaches to identify those with presynaptic defects in synaptic transmission. The advantage of this screen is that recessive lethal mutations can be recovered and studied because the eye is not required for fertility or viability. We have currently identified 32 different mutants with presynaptic defects in neurotransmitter release using this approach. These 32 mutations represent 14 complementation groups, of which at least nine also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. The neuronal fate determinant svp and the synaptic vesicle trafficking component lap were among the collection of mutants recovered in this screen.

Our other screening approach involves identification of modifiers of a rough eye phenotype resulting from overexpression of SNAP. The rationale for this screen is that the rough eye phenotype resulting from SNAP overexpression can be modified by altering the abundance of other secretory factors (see above). From a screen of 155 Deficiency bearing chromosomes that span most of the second and third chromosomes we identified 27 deficiencies that enhance and 26 deficiencies that suppress the SNAP overexpression phenotype. We are currently screening smaller overlapping deficiencies to narrow the intervals that contain these modifying loci and are testing P element insertions and known mutations that map to these intervals in an attempt to identify the relevant modifying genes. Future efforts will be directed at further characterization of the mutants recovered from classical screening approaches.

Our synaptic function studies are supported by the National Science Foundation.

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