InteractiveFly: GeneBrief

neuronal Synaptobrevin: Biological Overview | References


Gene name - neuronal Synaptobrevin

Synonyms - n-synaptobrevin

Cytological map position - 62A9-62A9

Function - membrane fusion v-SNARE

Keywords - vesicular protein, synaptic vesicle exocytosis, neurotransmitter release, endolysosomal trafficking, CNS, brain

Symbol - nSyb

FlyBase ID: FBgn0013342

Genetic map position - chr3L: 1631681-1637275

Classification - Synaptobrevin

Cellular location - vesicular transmembrane



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Ramirez, J., Martinez, A., Lectez, B., Lee, S. Y., Franco, M., Barrio, R., Dittmar, G. and Mayor, U. (2015). Proteomic analysis of the ubiquitin landscape in the Drosophila embryonic nervous system and the adult photoreceptor cells. PLoS One 10: e0139083. PubMed ID: 26460970
Summary:
Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Using an in vivo biotinylation strategy this study has isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, the proof-of-principle validation is reported of two lysine residues required for nSyb ubiquitination. These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described in this study complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.
Vasin, A., Volfson, D., Littleton, J. T. and Bykhovskaia, M. (2016). Interaction of the Complexin accessory helix with Synaptobrevin regulates spontaneous fusion. Biophys J 111: 1954-1964. PubMed ID: 27806277
Summary:
Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE; Syntaxin 1A) and vesicle-attached (v-SNARE; n-synaptobrevin) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. A model has been proposed in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs were combined in Drosophila. MD simulations of the Drosophila Cpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. Clamping properties were investigated in the syx3-69 paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. This mode was used to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. Transgenic Drosophila were generated with mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.

BIOLOGICAL OVERVIEW

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-Syb (neuronal Synaptobrevin) is a vesicular protein that plays a key role during synaptic vesicle exocytosis. This paper reports that loss of n-syb caused slow neurodegeneration independent of its well documented role in neurotransmitter release in adult Drosophila photoreceptor neurons. In addition to synaptic vesicles, n-Syb localizes to endosomal vesicles. Loss of n-syb lead to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. The evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescues n-syb-dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. These findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity (Haberman, 2012).

Soluble NSF attachment protein receptors (SNAREs) are the core proteins in membrane fusion. The neuron-specific synaptic v-SNARE n-syb (neuronal Synaptobrevin) plays a key role during synaptic vesicle exocytosis. This paper reports that loss of n-syb caused slow neurodegeneration independent of its role in neurotransmitter release in adult Drosophila photoreceptor neurons. In addition to synaptic vesicles, n-Syb localizes to endosomal vesicles. Loss of n-syb leads to endosomal accumulations, transmembrane protein degradation defects, and a secondary increase in autophagy. The evidence suggests a primary defect of impaired delivery of vesicles that contain degradation proteins, including the acidification-activated Cathepsin proteases and the neuron-specific proton pump and V0 adenosine triphosphatase component V100. Overexpressing V100 partially rescues n-syb-dependent degeneration through an acidification-independent endosomal sorting mechanism. Collectively, these findings reveal a role for n-Syb in a neuron-specific sort-and-degrade mechanism that protects neurons from degeneration. These findings further shed light on which intraneuronal compartments exhibit increased or decreased neurotoxicity (Haberman, 2012).

Regulated SNARE-mediated membrane fusion underlies a plethora of intracellular trafficking, sorting, and exocytosis and has been particularly well studied in neurotransmitter release. The synaptic v-SNARE n-Syb (neuronal Synaptobrevin)/VAMP2 is a key protein required for synaptic vesicle exocytosis. In Drosophila, n-Syb is a neuronal protein that functions partially redundantly with the ubiquitously expressed Cellubrevin (Bhattacharya, 2002). Thus, n-Syb represents one of several intracellular membrane trafficking proteins that serve specialized neuronal requirements for vesicle trafficking at the synapse (Haberman, 2012).

In addition to the synaptic vesicle cycle, neurons use specialized membrane trafficking and a neuronal intracellular degradation mechanism during development and adult maintenance. Loss of endolysosomal or autophagosomal degradation is sufficient to cause adult-onset degeneration in Drosophila photoreceptors and neurons in general. Autophagy and the canonical endolysosomal system are considered ubiquitous cellular membrane trafficking machinery that may serve specialized or increased functions in neurons. In addition to the ubiquitous degradation machinery, it has been recently reported that a neuronal vesicle ATPase (v-ATPase) component provides a neuron-specific intracellular degradation mechanism; loss of v100, the neuron-specific v-ATPase subunit a1, leads to defects in brain wiring (Williamson, 2010b) and adult-onset degeneration (Williamson, 2010a) in Drosophila. No other neuron-specific membrane trafficking protein required for intracellular degradation has so far been characterized (Haberman, 2012).

It has previously been reported that loss of n-syb in the Drosophila visual system leads to fine structural synaptic defects that have an onset before synapse formation (Hiesinger, 1999). Recent work has shown that v100 behaves similarly to n-syb. Specifically, loss of either protein leads to neurotransmitter release defects, and both proteins directly interact with the t-SNAREs Syntaxin and SNAP-25 at the synapse (Hiesinger, 2005). These observations give rise to the idea that both proteins might exert related functions in endolysosomal trafficking in addition to their roles in neurotransmitter release. This study shows that loss of n-syb causes intracellular degradation defects that lead to neurodegeneration in adult photoreceptor neurons. n-Syb functions in concert with V100 in a neuronal sort-and-degrade mechanism that is required for neuronal maintenance independent of their roles in neurotransmitter release (Haberman, 2012).

This paper has characterized an unexpected function of the neuronal v-SNARE n-Syb in endolysosomal trafficking that is independent of its known function in neurotransmitter release. Loss of this function leads to neurodegeneration in adult fly photoreceptor neurons. These findings explain earlier observations of late developmental defects during brain wiring that do not occur when neuronal activity or neurotransmitter release is impaired (Hiesinger, 1999, 2006). Genetic and cell biological evidence indicates that n-Syb functions in concert with the neuronal v-ATPase component V100 in a neuronal sort and degrade pathway. Specifically, the evidence supports a primary defect in the fusion of vesicles that contain degradation machinery (including degradation proteases and V100-containing ATPases required to activate them) with endosomal compartments (Haberman, 2012).

The ultrastructure of n-syb mutant terminals reveals accumulations of both small vesicles and large degradative and autophagosomal compartments. n-Syb is a small (<200 amino acids) protein that is very well characterized as a synaptic v-SNARE required for vesicle fusion (Broadie, 1995, Galli, 1995; Deitcher, 1998; Südhof, 2009). In addition, synaptic vesicles are sorted through synaptic endosomes. Hence, the most straightforward explanation for endolysosomal trafficking defects in n-syb mutant neurons would be a defect in vesicle fusion (Haberman, 2012).

Several observations indicate that the endolysosomal defect is not directly linked to n-Syb’s function in synaptic vesicle exocytosis. First, loss of neuronal activity or neurotransmitter release does not cause endolysosomal, developmental or degenerative phenotypes in Drosophila photoreceptors as shown in this study and previously (Hiesinger, 2006). Second, although vesicles accumulate, synaptic vesicle markers do not accumulate correspondingly. Instead, this study fpind that several markers of the endolysosomal system are up-regulated. In particular, the early endosomal t-SNARE Syx7, together with transmembrane receptors, including Chaoptin and Fasciclin 2, exhibits a strong up-regulation and evenly fills the profiles of enlarged synaptic terminals. Together with the EM data, it is concluded that synaptic terminals are filled with large numbers of endosomal vesicles rather than with synaptic vesicles (Haberman, 2012).

Several lines of evidence indicate that n-syb is not required for autophagosome maturation, at least up to a late stage. First, n-Syb colocalizes only little with autophagosomes but marks mostly endosomal compartments and synaptic vesicles. Second, autophagosomes are acidified and contain active proteases (Cathepsins) and degraded material based on EM, Atg8-mCherry-GFP measurements, and mature Cathepsin accumulations. Third, induction of autophagy by atg1 expression does not depend on n-syb. Finally, the accumulation of undegraded Chaoptin and Fasciclin 2 receptors that evenly fill terminals is not consistent with their accumulation in autophagosomes. Because autophagy typically initiates in response to nutrient deprivation or other neurotoxic insults, the appearance of autophagosomes may be a secondary response (Haberman, 2012).

What then is n-Syb's other vesicle fusion role? Evidence points to at least two types of small vesicles accumulating in n-syb terminals: small endosomal vesicles and Cathepsin-containing ER/Golgi-derived transport vesicles. The early accumulation of pro-Cathepsin-containing vesicles suggests a defect in vesicle fusion and is not easily explained by defective exocytosis at the plasma membrane. Importantly, observations show that most intracellular vesicle fusion events are unaffected by loss of n-syb, consistent with the presence and partially redundant function of the ubiquitous v-SNARE Cellubrevin in photoreceptors (Bhattacharya, 2002). Most prominently, the complete absence of any early developmental defect implies that n-syb cannot be required for the canonical secretory pathway that regulates morphogen and receptor exocytosis. Similarly, spatiotemporally regulated receptor endocytosis appears unaffected because receptor accumulations distribute evenly in the enlarged n-syb mutant terminals. It is concluded that receptors accumulate in small endocytic vesicles downstream of endocytosis and upstream of further sorting or degradation. Collectively, these observations pinpoint a role for n-Syb in the fusion of Cathepsin/V100-containing (degradation machinery) vesicles with to-be-degraded compartments. The latter do not include autophagosomes, as Cathepsin delivery to autophagosomes is apparently unimpaired. Furthermore, the large numbers of Syx7-positive small endosomal vesicles are not likely a fusion partner because Syx7 does not directly interact with n-Syb in this study and in vertebrates (Antonin, 2000). Instead, the function cam be narrowed down to a fusion reaction between V100/Cathepsin degradation machinery vesicles with neuronal endolysosomal compartments before the formation of lysosomes. The n-Syb-mediated endolysosomal fusion event could be directly between degradation machinery vesicles and larger or intermediate endosomal vesicles or sorting compartments, with which fusion of small endosomal vesicles is also blocked in the absence of n-syb. In either case, the intravesicular endosomal fusion uncovered by loss of n-syb represents a neuronal specialization and possible addition to the ubiquitous endolysosomal machinery (Haberman, 2012).

It has been recently reported that the neuron-specific v-ATPase component V100 defines a neuronal sort-and-degrade mechanism that increases neuronal degradative capacity (Williamson, 2010c; Williamson, 2010a). Loss of n-syb and v100 reveals striking similarities with one remarkable difference: endosomal accumulations in n-syb are considerably more severe and lead to a several-fold enlargement of synaptic terminals and yet a significantly slower progression of degeneration than in v100. This difference is exacerbated by overexpressing v100 in n-syb mutant neurons, leading to yet more endosomal cargo colocalization, while further ameliorating the degeneration. The genetic and cell biological interactions indicate that n-syb and v100 function in concert in a neuronal sort-and-degrade mechanism. The inverse correlation between endosomal accumulations and neuronal degeneration further suggests that the endosomal accumulations are not per se neurotoxic and less toxic than accumulations in late degradative compartments (Haberman, 2012).

What then kills n-syb mutant neurons? Two main mechanisms are thought to underlie cell death in most neurodegenerative conditions. First, the neuron may become metabolically compromised when amino acids and other molecules are not recycled and accumulate in undegraded compartments; second, the accumulations themselves may be toxic. The latter hypothesis is typically discussed for accumulations in late degradative compartments, as they have a highly toxic content in terms of pH and proteases (leaky lysosomes). This idea is consistent with the observation of reduced toxicity through increased accumulations in early endosomal compartments in n-syb and further with v100 overexpression in the n-syb mutant. It is therefore speculated that the appearance of more neurotoxic late degradative compartments is the likely cause of death in n-syb mutant photoreceptors (Haberman, 2012).

Notably, both V100 and n-Syb are synaptic proteins that have first been characterized as components of synaptic vesicles. The finding that both proteins additionally function in neuronal maintenance through an endolysosomal sort-and-degrade mechanism suggests that the synaptic vesicle cycle may be interlinked with v100- and n-syb-mediated endosomal trafficking. Furthermore, these observations suggest the resulting maintenance function predominantly operates at synapses. It is therefore proposed that v100 and n-syb mutants reveal a neuron-specific synaptic extension of the endolysosomal system that serves the specialized and extensive demands of neurons, and of synapses in particular, on intracellular membrane trafficking (Haberman, 2012).

Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses

Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, the fluorescence responses were measured from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach it was found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission (Sinha, 2011).

In conventional neuronal synapses, fast synaptic transmission is mediated by release of neurotransmitter upon Ca2+-triggered synaptic vesicle (SV) exocytosis. This process is exquisitely regulated both spatially and temporally. The core of the SV fusion machinery is formed by three members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein family, which is characterized by conserved sequences of 60-70 amino acids called SNARE motifs: vesicle SNARE protein, synaptobrevin-2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25 (Sinha, 2011).

Zipper-like assembly of the SNARE motifs from their N-terminal ends toward their membrane-proximal C termini results in the formation of a highly stable heterotrimeric 'trans-SNARE complex' (also called 'SNAREpin'), consisting of four parallel α-helices, which brings the two membranes into close apposition for fusion. Previous studies have suggested that several of these SNARE complexes might assemble in rosette-shaped multivalent supercomplexes, forming a ring, around the fusion pore; however, there is no direct evidence in support of this model. Therefore, the precise number of SNARE complexes minimally required to drive membrane fusion is highly debated and current estimates range between 1 and 15. Some of these results are based on single-molecule fluorescence measurements in artificially reconstituted liposomes, whereas others are based on theoretical models, kinetic analysis, and extrapolations from dose-response relationships. Therefore, it is essential to apply a more direct method capable of visualizing single SNARE complexes in real time in a physiological setting (Sinha, 2011).

This study used a unique direct approach to count the number of syb2 molecules required for fast Ca2+-triggered exocytosis in living hippocampal neurons. To quantify the effective number of syb2 molecules, the fluorescence responses were measured from single fusing vesicles expressing synaptopHluorin (spH) on a genetic null background and calibrated the fluorescence signals to those of single pHluorin (pHl) molecule fluorescence measured in vitro (Sinha, 2011).

This study has resolved the minimum number of syb2 required for executing SNARE-dependent membrane fusion in functionally intact synapses. Using high-resolution fluorescence measurements of single-vesicle fusion followed by calibration with single-molecule measurements the exact number of pHl-tagged vesicle proteins inserted per SV. was counted. The δF distributions are quantized where single pHl molecules were resolved as distinct peaks of mean size equivalent to that estimated from single pHl molecule measurements in vitro. However, when spH was overexpressed in syb2/ceb2 DKO boutons, the δF distributions exhibited a dramatic absence of the first molecular peak obtained from single-vesicle spH fluorescence measurements in DKO neurons, which clearly defined the lower bound of 2 syb2/spH molecules required to evoke fusion during fast synaptic transmission (Sinha, 2011).

These findings provide several insights into the process of vesicle docking, priming, and fusion during fast synaptic transmission. With a minimum of two spH molecules per SV it is difficult to imagine that SVs dock with their two SNAREs already pointing to the plasma membrane. Rather the two SV SNAREs should freely diffuse within the SV membrane and therefore should be positioned randomly on the SV surface during docking. Thus, these findings imply that initial docking is syb2 independent but rather driven by other factors such as Munc18-1, syntaxin-1, or, as recently shown, synaptotagmin-1 (Sinha, 2011).

During regulated exocytosis, merger of the two membranes leads to the formation of an aqueous fusion pore whose physical properties have been long debated. On the basis of the current results it is suggested that the fusion pore is likely to be composed of lipids, two transmembrane domains of syb2, and two of syntaxin 1A. This suggestion implies that the inner fusion pore is predominantly lined by lipids instead of transmembrane SNARE domains arranged like barrel staves around the pore (Sinha, 2011).

The finding that such a low copy number of syb2 can rescue evoked fusion raises the possibility that the kinetics of synaptic transmission, observed using spH overexpression in DKO neurons, may be slower than during normal physiological neurosecretion when there are 70 copies of syb2 present on the SV. However, an earlier as well as a recent study have shown that overexpression of N- or C-terminal GFP fusion constructs of syb2 in syb2-deficient hippocampal neurons fully rescues the amplitude and kinetics of evoked excitatory postsynaptic potentials in syb2-deficient neurons (Sinha, 2011).

SNARE assembly is believed to generate sufficient energy to drive membrane fusion. Recent studies using a surface force apparatus indicated that the stabilization energy of a single partially assembled neuronal SNARE complex is ~35 kBT, which corresponds closely to the energy required for hemifusion of lipid bilayers (40-50 kBT). Moreover, using isothermal titration calorimetry, the free energy estimated for the assembly of individual SNARE complexes was found to be sufficient for membrane fusion. Thus, assembly of one SNARE complex could in theory drive fusion. Indeed, a recent study based on in vitro Förster resonance energy transfer experiments indicates that liposomes bearing only a single SNARE molecule are still capable of fusion with other liposomes or purified SVs. Why in vivo more SNARE complexes are needed than in vitro remains to be elucidated. One reason might be the different timescales on which SV fusion and in vitro fusion proceed: Whereas AP-triggered SV fusion in the current experiments is completed within milliseconds, in vitro fusion takes seconds, indicating another very slow rate-limiting step upstream of SNARE complex formation in vitro. A trivial reason might be that syb2 is trafficked to the synapse and hence SVs as dimers. In this case, however, suppression not only of the first but also of the third peak in the amplitude histogram of spH expressing DKO neurons would be expected, which was not observed. Finally, it needs to be borne in mind that in the reconstitution experiments SNAREs were fully active and not complexed to any of the control proteins such as synaptotagmin, Munc18, or complexin, whose binding in turn lowers the total energy that becomes available for fusion during SNARE assembly. In conclusion, the current finding that two syb2 molecules and likely two SNARE complexes are sufficient and necessary for SV priming and fast Ca2+-triggered exocytosis fundamentally revises the understanding of SNARE-mediated fusion pore formation and membrane fusion (Sinha, 2011).

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila

The V0 complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. Mutations were isolated in the largest V0 component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is required only in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Both GST-V100 and GST-Syx retrieve His-tagged SNAP-25. Furthermore, GST-V100 also interacts with His-Syx. In contrast, only GST-Syx, but not GST-V100, pulls down His-n-Syb, indicating that V100 directly interacts with Syx and SNAP-25, the SNAREs at the target membrane, but not with the vesicle-SNARE n-Syb. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. The data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion (Hiesinger, 2005).

Vesicular or vacuolar ATPases are the most prominent intracellular proton pumps, consisting of at least 12 subunits in two sectors (V0 and V1). Acidification is important for many cellular functions, including receptor-ligand dissociation, degradative pathways, and the generation of intercompartment proton motive forces that are in turn utilized as driving forces for numerous secondary transport processes. This study reports the consequences of the selective disruption of a V0 subunit a1 homolog in neurons. The V0 subunit a is encoded by four homologous genes in flies, worms, mouse, and human. The data from yeast and C. elegans indicate a crucial role of V0 subunit a proteins for specific functions in distinct intracellular compartments and different cell types (Hiesinger, 2005).

Mutations were isolated in v100 based on the specific defect of photoreceptor neurons to evoke a postsynaptic response. Photoreceptors are an excellent 'test tube' because they are not required for viability of the fly, and numerous assays can be used to assess morphology and function. Since loss of v100 does not affect photoreceptor specification, development, viability, and the ability to sense light, it is surmised that most intracellular vesicle trafficking and acidification processes are unaffected. Indeed, mutations that affect two key protein components of the V1 subcomplex (subunits A and B) are cell lethal when removed in photoreceptors, and acidification as measured with LysoSensor in v100 mutant photoreceptor cell bodies is unaffected. Hence, if acidification is the cause of the observed phenotypes, it is likely to only affect synaptic vesicles (Hiesinger, 2005).

Several methods can be used to directly or indirectly assess the acidification of synaptic vesicles. The pH-sensitive dye LysoSensor or genetically encoded pHluorin, a pH-sensitive GFP fusion protein that localizes within vesicles, can be used in Drosophila neurons to directly assess synaptic vesicle acidification. Unfortunately, the intensity differences at embryonic NMJs are too small to be observed with the confocal system used. However, several lines of evidence allow an assessment of the contribution of a possible acidification defect to the observed phenotypes. Several results are not consistent with a defect in synaptic vesicle acidification alone. These include (1) the accumulation of vesicles in mutant terminals, (2) the normal mEPSC amplitude and frequency combined with a severely reduced evoked response in a hypomorphic allelic combination, and (3) the impairment of FM1-43 uptake. In addition, several lines of evidence are not readily explained with the function of V100 as part of a proton pump: (1) selective partial rescue of vesicle trafficking but not acidification in yeast, (2) selective interaction with t-SNAREs, and (3) localization at active zones. Taken together, these results indicate that V100 exerts additional or alternative functions to synaptic vesicle acidification at Drosophila synapses (Hiesinger, 2005).

There is wide support for the hypothesis that SNAREs form the basic molecular apparatus that forces lipid bilayers to fuse. However, this does not imply that SNAREs alone are required to induce synaptic vesicle exocytosis. SNAREs alone are sufficient to induce fusion of liposomes, but the kinetics of these events does not mimic the kinetics that has been observed in synaptic vesicle fusion in vivo. The yeast vacuolar fusion assay is the only system in which evidence for an additional component downstream of SNARE function has been identified. Interestingly, the isolation of a proteolipid pore complex from synaptosomes from electroplaques of Torpedo, named the 'mediatophore' has been isolated. This pore complex contains a subunit of the V0 complex and transfection of this component in some cells allows quantal release of neurotransmitter. Subunit a1 localizes to synaptic terminals and interacts with the v-SNARE n-Syb. While the localization is in agreement with the current findings at Drosophila synapses, selective interactions of V100 with the t-SNAREs Syx and SNAP-25 were found in agreement with the observations made in yeast (Hiesinger, 2005).

These analyses have revealed many similarities between v100 and n-syb mutants: both die as late embryos without coordinated movement, accumulate vesicles at synapses, exhibit reduced spontaneous vesicle release and FM1-43 uptake, contain vesicles with normal transmitter content that is poorly released upon stimulation, and interact with t-SNAREs. The only assay in which n-syb mutants behaved differently from v100 is the hypertonic stimulation. Vesicle release induced with hypertonic solution in Drosophila SNARE mutants is largely abolished. In contrast, v100 mutants exhibit some responses with a reduced number of events but normal amplitude. This implies (1) the presence of neurotransmitter-loaded vesicles in mutant terminals and (2) that SNARE function required for hypertonic release is at least partially possible. V100 is not crucial for this step, placing its role downstream of SNARE-dependent priming, congruous with the findings in yeast vacuolar fusion. A function downstream of priming is also supported by the observation that Syx overexpression phenotypes are suppressed by the loss of v100 (Hiesinger, 2005).

The results show only a late exocytic role for the V0 subunit a1, while the implication of other V0 components remains to be tested. Hence, the data is formally consistent with a role of the V0 subunit a1 outside of the V0 complex in association with SNAREs. However, no role outside the V0 complex has so far been shown for a subunit a in any system. In summary, the data indicate a function for V100 and possibly the V0 proteolipid pore as a mediator of vesicle release efficiency downstream of SNARE-dependent priming (Hiesinger, 2005).

Absence of junctional glutamate receptor clusters in Drosophila mutants lacking spontaneous transmitter release

Little is known about the functional significance of spontaneous miniature synaptic potentials, which are the result of vesicular exocytosis at nerve terminals. By using Drosophila mutants with specific defects in presynaptic function it has been found that glutamate receptors cluster normally at neuromuscular junctions of mutants that retain spontaneous transmitter secretion but have lost the ability to release transmitter in response to action potentials. In contrast, receptor clustering is defective in mutants in which both spontaneous and evoked vesicle exocytosis are absent. Thus, spontaneous vesicle exocytosis appears to be tightly linked to the clustering of glutamate receptors during development (Saitoe, 2001).

The existence of miniature end-plate potentials provides a basis for the theory of quantal synaptic transmission. A single miniature end-plate potential arises when a synaptic vesicle fuses spontaneously with the presynaptic membrane and releases a quantum of transmitter (spontaneous vesicle exocytosis). However, little is known about the functional importance of this process. Presynaptic and postsynaptic neurotoxins that allow spontaneous vesicle exocytosis to persist have little effect on synaptic development, including postsynaptic accumulation of receptors. During the development of Drosophila neuromuscular junctions (NMJs), glutamate receptors (GluRs) cluster in the postsynaptic membrane in a manner that depends on nerve-muscle contact. To investigate the role of spontaneous secretory events in receptor clustering, Drosophila mutants with distinctive secretory defects were used. Mutations of neuronal-synaptobrevin (n-syb) or cysteine string protein (csp) selectively prevent nerve-evoked exocytosis whereas spontaneous vesicle exocytosis persists. In contrast, syntaxin-1A (syx) or shibire (shi) mutations eliminate both spontaneous and evoked exocytosis, thereby allowing one to distinguish the role of spontaneous secretory events (Saitoe, 2001).

Neuromuscular transmission in wild-type and mutant Drosophila embryos or larvae were characterized. A typical burst of excitatory synaptic currents (ESCs) often exceeded 600 pA in amplitude in a newly hatched wild-type larva (control). In the presence of tetrodotoxin, the bursting of ESCs is suppressed, and ESCs seldom exceeded 400 pA. A similar suppression of bursting and reduction in the amplitude of ESCs is observed when the ventral nerve cord is removed. Thus, propagated activity in the nervous system triggers multiple vesicle exocytosis and contributes to the ESCs. Concomitantly, the residual events [miniature ESCs (mESCs)] in these wild-type larvae are due to spontaneous vesicle exocytosis (Saitoe, 2001).

An n-syb null mutant was investigated in which nerve-evoked ESCs but not mESCs are lost. Consistent with these findings, ESCs were detected in n-syb embryos but virtually no large-amplitude ESCs characteristic of nerve-evoked ESCs. This apparent absence of evoked responses (but persistence of mESCs) was confirmed by the fact that TTX had no effect on the frequency or amplitude of ESCs and that no evoked ESCs were elicited by nerve stimulation (Saitoe, 2001).

In syx mutants, both nerve-evoked and mESCs are undetectable. In agreement with this phenotype, neither nerve-evoked nor mESCs were detected during observations exceeding 15 min each in seven cells. Although the possibility of missing very infrequent occurrences could not be completely eliminated, it is clear that the frequency of mESCs in syx embryos is far lower than the frequency of mESCs in n-syb embryos. Given the distinct phenotypes of the n-syb and syx mutants, the distribution of postsynaptic GluRs was examined in these embryos (Saitoe, 2001).

Preparations were also stained with antibody against horseradish peroxidase (anti-HRP), which binds to a neuronal surface antigen and reveals the presynaptic terminals. Immunoreactive GluRs formed prominent junctional clusters that closely mirrored the presynaptic elements in wild-type and n-syb mutants. Although this finding apparently contrasts with the observation in para (Na+ channel) mutants, that neural electrical activity is essential for the clustering of receptors, it should be noted that the n-syb mutation is a more subtle perturbation of this system. Unlike n-syb, however, syx mutants rarely have discernible junctional GluR clusters, although they invariably form neuromuscular contacts (Saitoe, 2001).

These findings raised the possibility that the absence of detectable mESCs in these mutants may have arisen from a deficit of postsynaptic GluRs. Moreover, the lack of receptor clusters could either be a developmental consequence of a lack of vesicle fusions in the nerve terminal, or could be due to a requirement for syntaxin in the trafficking of GluRs to the postsynaptic membrane. Indeed, syx is required for cell viability in Drosophila, and the development of both the neuron and muscle in syx embryos is likely to be due to small amounts of maternal Syx. If this residual Syx is not adequate for the maintenance of normal vesicular traffic to the cell surface, GluRs may not be inserted appropriately in the sarcolemma. To address these issues, it was determined whether syx mutants responded to applied glutamate and also whether junctional GluR clusters are restored in syx mutants by selectively inducing the presynaptic or postsynaptic expression of a syx transgene (Saitoe, 2001).

The application of L-glutamate by pressure ejection onto the junctional region of syx muscles indicates that some sensitivity is lost concomitantly with the loss of clusters. The pressure ejection of L-glutamate at the junctional region yielded robust inward currents in wild-type and n-syb mutants, but glutamate-evoked currents are much smaller in syx mutants. What is important here is that even with this diminished glutamate sensitivity, mESCs would have been detected in syx mutants if their terminals were spontaneously releasing transmitter. Thus, the complete absence of detectable mESCs could not be attributed to a lack of postsynaptic sensitivity (Saitoe, 2001).

The low sensitivity to glutamate in syx mutants could reflect the diminished trafficking of GluRs to the surface. Thus, a test was performed to see whether the clustering defect is due to a pre- or post-synaptic action of syx by the targeted expression of the syx transgene with either the neuron-specific elav-GAL4 driver or the muscle-specific G14-GAL4 driver. Neuron-specific expression of the syx transgene restores GluR clusters but muscle-specific expression does not. Evoked and mESCs are readily observed in transgenic embryos in which neuron-specific expression of the syx transgene is restored but not in transgenic embryos in which syx transgene is expressed in muscles. Thus, a comparison of the phenotypes of n-syb and syx has led to a hypothesis that spontaneous secretory events at the NMJ are critical to the formation of GluR clusters (Saitoe, 2001).

Two temperature-sensitive (ts) paralytic mutants were used to examine further the correlation between spontaneous vesicle exocytosis and GluR clustering. At elevated temperatures, a defect in dynamin in shits blocks synaptic vesicle recycling and thereby depletes the terminals of synaptic vesicles. In contrast, cspts mutations appear to interfere with excitation-secretion coupling in the terminal. Synapses in shits mutants become completely silent at a nonpermissive temperature (32°C), whereas cspts mutants lose evoked responses while retaining mESCs. Because of these differences in release properties at the nonpermissive temperature, the distribution of GluRs in these lines was compared. At a permissive temperature (25°C), when release properties are similar among these embryos, GluR clustering is comparable for wild-type, cspts, and shits mutant embryos. However, this situation changes when embryos are moved to the nonpermissive temperature at 13 hours after fertilization, which is when nerve-muscle contacts first form. The development of GluR clusters is not perceptibly altered in wild-type and cspts mutants at 32°C. However, no detectable GluR clusters are observed in shits mutants, as is the case in syx mutants. These results again suggest a tight link between spontaneous vesicle exocytosis and GluR clustering (Saitoe, 2001).

Further insight into the nature of interaction of presynaptic and postsynaptic elements has come from the injection of argiotoxin into wild-type embryos at concentrations that block all muscle contractile activity. In these embryos, GluRs still cluster postsynaptically. Thus, it was not the activation of postsynaptic GluRs that directs GluR clustering. Similar findings have been reported in vertebrates, where alpha-bungarotoxin does not impede the clustering of acetylcholine receptor (AChR). As in vertebrates, secretion of molecules, such as agrin for AChRs, ephrins for N-methyl-D-aspartate (NMDA)-type GluRs, and neuronal activity-regulated pentraxin for AMPA-type GluRs, may drive receptor clustering by being released with, or in parallel to, the neurotransmitter at Drosophila NMJs (Saitoe, 2001).

A positive correlation has been documented between ongoing spontaneous vesicle exocytosis and the embryonic development of GluR clusters at Drosophila NMJs. Nerve-evoked vesicle exocytosis is not necessary for this process, because although neither n-syb nor cspts mutants show any demonstrable nerve-evoked ESCs, GluRs still cluster. mECSs persist in both mutants. However, when spontaneous secretory events are absent (as in shits mutants at the nonpermissive temperature or in syx), junctional GluR clusters are exceedingly infrequent. If GluR clustering is solely contingent on the nerve-muscle contact, GluRs should cluster at the contacts in shits mutants raised at the nonpermissive temperature and in syx mutants. In a recent study of mice lacking an isoform of munc 18-1, there was no demonstrable change in AChR clustering, although both spontaneous and evoked neurotransmitter release are absent. Together with the observations made in this paper, these data suggest that munc 18-1 is not involved in the secretion of the agent that induces clustering of neurotransmitter receptors, whereas syntaxin is essential for this process (Saitoe, 2001).

The absence of clusters in Drosophila in syx and shi mutants implies that spontaneous secretory events are related to GluR clustering and probably to cluster stabilization as well. Moreover, it is the clustering of these receptors, rather than their surface expression, that depends on spontaneous secretion: Functional GluRs are detected in syx mutants although they rarely form detectable clusters at the synapse. The link between spontaneous vesicle exocytosis and receptor clustering must now be clarified (Saitoe, 2001).

Two independent pathways mediated by cAMP and protein kinase A enhance spontaneous transmitter release at Drosophila neuromuscular junctions

cAMP is thought to be involved in learning process and known to enhance transmitter release in various systems. In two Drosophila memory mutants, dunce and rutabaga, the cAMP cascade is defective, and no post-tetanic facilitation is observed at the neuromuscular junction. Thus, changes in synaptic efficacy were suggested for the molecular mechanism of memory. In Aplysia, cAMP mediates changes in synaptic transmission during dishabituation, sensitization, and classical conditioning. cAMP blocks various types of K+ channels, which in turn leads to membrane depolarization and/or a prolongation of presynaptic action potentials, and finally results in an activation of voltage-gated Ca2+ channels. The long-term potentiation (LTP) at the bullfrog sympathetic ganglion and at the rat hippocampal CA3 is also mediated by cAMP and requires Ca2+ influx at presynaptic terminals. In other cases, cAMP directly enhances Ca2+ influx through modulation of Ca2+ channels. The Ca2+-independent effect of cAMP has also been demonstrated in the crayfish neuromuscular junction and in cultured mammalian CNS neurons. Thus, the effects of cAMP on synaptic transmission are diverse. Multiple mechanisms might be operating in parallel in one synapse (Yoshihara, 2000).

Using Drosophila genetics it is possible to separate the multiple mechanisms involved in the effects of cAMP on synaptic transmission. Synaptic transmission has been examined in Drosophila embryos lacking a synaptic vesicle protein, neuronal-synaptobrevin (n-syb), which is a v-soluble NSF attachment protein receptor (SNARE) protein, and required for nerve-evoked transmitter release. Even though evoked release is absent, miniature synaptic currents (mSCs) are readily observed in n-syb null mutants. Their frequency increases in response to an increase of Ca2+ concentrations in high K+ saline. A Ca2+ ionophore, A23187, also increases the mSC frequency in the presence of external Ca2+. These findings indicate that the n-syb null mutants are still capable of responding to elevations of internal Ca2+. Furthermore, in wild-type embryos cAMP increases the frequency of mSCs in the absence of external Ca2+, but does not in the n-syb null mutants. Thus, requirements for two modes of vesicle fusion, spontaneous transmitter release and nerve-evoked release, seem to be different (Yoshihara, 2000).

The preceding results, showing that in the absence of external Ca2+ cAMP enhances spontaneous transmitter release, suggest two basic features regarding the effects of cAMP on spontaneous transmitter release. (1) This pathway involves n-syb, a protein that is essential for evoked release (n-syb-dependent pathway). (2) cAMP enhancement of release is not dependent on external Ca2+. It has been asked, using the n-syb null mutant, whether cAMP also enhances spontaneous transmitter release through an increase of intracellular Ca2+ when Ca2+ is available. Further is has been asked whether PKA encoded by DC0 is involved in this enhancing effect of cAMP on transmitter release (Yoshihara, 2000).

cAMP has been shown to enhance spontaneous transmitter release in the absence of extracellular Ca2+ and n-syb is required in this enhancement (n-syb-dependent). The cAMP-induced enhancement of transmitter release was examined in the presence of external Ca2+. The intracellular concentration of cAMP was raised by application of either forskolin, an activator of adenylyl cyclase, or by 4-chlorophenylthio-(CPT)-cAMP, a membrane-permeable analog of cAMP, in the presence of external Ca2+, while recording miniature synaptic currents (mSCs) at the neuromuscular junction in n-syb null mutant embryos. The frequency of mSCs increases in response to elevation of cAMP, and this effect of cAMP is completely blocked by Co2+ (n-syb-independent pathway). In contrast, in wild-type embryos the cAMP-induced mSC frequency increase is partially blocked by Co2+. In DC0, a mutant defective in protein kinase A (PKA), nerve-evoked synaptic currents are indistinguishable from the control, but mSCs are less frequent. In this mutant the enhancement by cAMP of both nerve-evoked and spontaneous transmitter release is completely absent, even in the presence of external Ca2+. Taken together, these results suggest that cAMP enhances spontaneous transmitter release by increasing Ca2+ influx (n-syb-independent) as well as by modulating the release mechanism without Ca2+ influx (n-syb-dependent) in wild-type embryos, and these two effects are mediated by PKA encoded by the DC0 gene (Yoshihara, 2000).

In a DC0 mutant, the amplitude and Ca2+ dependency of nerve-evoked synaptic currents are not significantly different from those in wild-type, whereas the mSC frequency is lower. Conversely, in a n-syb null mutant, n-syb DeltaF33B, no nerve-evoked synaptic currents are detected, whereas mSCs are readily observable. Thus, it appears that these two modes of vesicle fusion, nerve-evoked and spontaneous, seem to have distinct requirements (Yoshihara, 2000).

Under various conditions there is a good correlation between the frequency of mSCs and the number of quanta released by nerve stimulation. In rat cerebellar synapses, there is a clear correlation between the frequency of mSCs and the amplitude of evoked synaptic currents in preparations treated with various concentrations of forskolin. In accordance with this, in wild-type embryos, forskolin increases the frequency of mSCs and the quantal content in a similar time course. Furthermore, in the DC0 mutant, the effect of forskolin is observed neither in spontaneous transmitter release nor in nerve-evoked release. These results suggest that these two modes of transmitter release are similarly affected by cAMP-PKA (Yoshihara, 2000).

cAMP is shown at the Drosophila neuromuscular junction in third instar larvae to increase the size of exo-endo cycling pool (readily releasable pool) of synaptic vesicles. The size of this pool is closely correlated with the quantal content of synaptic potentials evoked by nerve stimulation at a low frequency. Forskolin increases the mSC frequency in wild-type embryos and in newly hatched wild-type larvae. Thus, it is likely that this pool supplies vesicles for both modes (nerve-evoked and spontaneous) of transmitter release. However, the two modes dichotomize after this step. For nerve-evoked release n-syb protein is required, whereas for spontaneous fusion this protein is not of absolute necessity, although its presence facilitates spontaneous transmitter release. The cAMP-PKA cascade seems to influence the vesicle fusion process at multiple levels: (1) vesicle mobilization and translocation, which increase the size of exo-endo cycling pool; (2) modification of Ca2+ influx through voltage-gated Ca2+ channels (n-syb-independent pathway), and (3) modulation of transmitter vesicle fusion (n-syb-dependent pathway). It is likely that the first mechanism affects both modes of vesicle fusion similarly. However, the second and third mechanisms may act differentially on the two modes of vesicle fusion, which may explain the phenotype of the DC0 mutant, namely at the resting state the cAMP-PKA cascade might not be affecting the nerve-evoked transmitter release, whereas spontaneous release might be supported by the baseline activity level of the cascade (Yoshihara, 2000).

Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on Synaptobrevin

To investigate a possible involvement of synaptic machinery in Drosophila visual system development, the effects of a loss of function of neuronal synaptobrevin (n-syb), a protein required for synaptic vesicle release, were studied. Expression of tetanus toxin light chain (which cleaves neuronal synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. Targeted tetanus toxin light chain (TeTxLC) expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. These results demonstrate the requirement of neuronal synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link to fine-tuning processes that may include synaptic plasticity in the development of the Drosophila CNS is discussed (Hiesinger, 1999).

The finding of an onset of n-syb expression in the first half of pupation poses the question of whether synapses actually start to function so early during optic lobe development. Neuronal activity plays a major role during vertebrate visual system development. A critical period of 1 d after eclosion has been demonstrated for experience-dependent developmental plasticity in the Drosophila. It has not yet been shown whether synaptic plasticity in the Drosophila CNS extends to pupation or whether neurotransmitters are released before any form of neuronal activity. Assuming the involvement of such processes, the following time scale would be expected: first, expression and localization of proteins of the vesicle release machinery; second, release of neurotransmitter independent or dependent on spontaneous activity; and third, release of neurotransmitter dependent on evoked activity. Given the early immunoreactivity of specific synaptic vesicle cycle proteins such as n-syb and synaptotagmin before P + 25% (25% through the pupal period), the synaptic vesicle cycle appears to be available for more than half of pupal development before first evoked photoreceptor responses occur at P + 82%. Morphological analysis has revealed a brief interval of intense synapse formation in the lamina of Musca starting ~P + 62% and peaking at P + 74%. Although this time window does not necessarily correspond to the first occurrence of synapses in the optic lobe of Drosophila, and the heterogeneity of optic lobe neurons should also be taken into consideration, it may indicate that n-syb is expressed long before synapses are morphologically recognizable (Hiesinger, 1999 and references).

Apparently, not all processes between target selection and the establishment of functional connectivity are yet known. The demonstration of the dependence of neuropil patterning on NO release shows a process of terminal development in a similar time window as the neuropil patterning defects observed in the current study. With regard to the current study, one possibility would be the involvement of n-syb in the release of neurotransmitters or other factors before or during synapse development. Li (1997) observed that histamine is synthesized in photoreceptors extending from cultured imaginal disks. Histamine or other substances released by growth cones after arrival in their target layers might exert functions necessary for the establishment of a regular terminal pattern (Hiesinger, 1999).

Lack of functional n-syb has no obvious influence on target selection and the development of largely overlapping terminals of R7 and R8 cells. In contrast, further development of terminal fine structure between P + 25 and P + 50% is significantly disturbed, indicating its involvement in a fine-tuning process. This early onset of n-syb function shows that either n-syb is involved in nonsynaptic processes taking place soon after target recognition, or synapses form earlier in the Drosophila optic lobe than is generally believed. Because this time window occurs significantly before the observed upregulation of Fasciclin II in active toxin-expressing photoreceptors, the morphological changes do not depend on this CAM (Hiesinger, 1999).

Cell adhesion molecules play multiple roles during optic lobe development. Most fruitfully investigated are functions during axon guidance and target recognition and synaptic plasticity. The finding of increased Fasciclin II immunoreactivity under conditions of blocked neurotransmitter release corresponds to previous studies that have shown the opposite effect with an opposite approach: apCAM is downregulated after application of serotonin, and synaptic Fasciclin II is reduced in mutants with abnormally high neuronal activity. Although it has been demonstrated that apCAM is downregulated via endocytosis, the mechanism of activity-dependent Fasciclin II downregulation at the Drosophila neuromuscular junction remains unknown. Possible downregulation mechanisms to be considered include endocytosis, extracellular cleavage, and reduced transcription or translation in combination with a continuous turnover of the protein. The upregulation of two different types of CAMs (Fasciclin II and chaoptin) in the same cell type under conditions of blocked neurotransmitter release poses the question of the specificity of the mechanism. In the absence of functional n-syb, increased numbers of docked synaptic vesicles accumulate presynaptically. Assuming that this would result in a significant sequestration of membrane material and that the synaptic vesicle cycle is continuously replenished from cell surfaces carrying adhesion molecules, deactivation of n-syb could result in decreased intake of CAMs and thus increased CAM immunoreactivity. However, current understanding of the recycling mechanism in the synaptic vesicle cycle and different localization of CAM isoforms does not support this hypothesis. Alternatively, specifically CAMs on active terminals and fibers could be downregulated to serve as markers for the competence of the synapses for sprouting (Hiesinger, 1999).

In wild-type third instar larvae, Fasciclin II is found on R7 and R8 retinal axons. During parts of pupation, Fasciclin II is detectable at low levels on photoreceptor cell bodies. It is possible that Fasciclin II is never completely downregulated from R7 and R8 terminals but is mostly below threshold for visualization with confocal microscopy. Upregulation of Fasciclin II levels in photoreceptors lacking functional n-syb after P + 75% may thus be attributable to an accumulation of the protein, when its downregulation would normally occur via an n-syb-dependent mechanism as part of a continuous protein turnover. The finding that IrreC-rst immunoreactivity remains unaltered in photoreceptors without functional n-syb but is increased in proximal neuropils after widespread TeTxLC expression can be interpreted in two different ways: either IrreC-rst protein is not present on photoreceptor terminals at the addressed time of pupation, or the n-syb-dependent CAM downregulation mechanism has a different molecular specificity in photoreceptors than in other optic lobe cells. During axonal pathfinding IrreC-rst is expressed on photoreceptors. In pupal stages IrreC-rst is localized to rhabdomeres but not to axons and cell bodies of photoreceptors during the second half of pupation. Because rhabdomeres are unique to this cell type and seem to be a preferred localization for IrreC-rst in photoreceptors, a cell-specific distribution that excludes terminals appears more likely than a specific CAM regulation mechanism for photoreceptors. Taken together, the results presented here clearly show a requirement of n-syb for optic lobe development. Either n-syb has a previously unknown activity-independent function, or synaptic transmission is involved in optic lobe development, or both (Hiesinger, 1999 and references).

Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on synaptobrevin

Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To further characterize the defects in these mutants, an examination was carried out of conditions that stimulate secretion. Despite the inability of an action potential to trigger fusion, high K+ saline can increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner: the rate of fusion approaches 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants can be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to sustained increases in cytosolic Ca2+ persists in the absence of this protein. Tetanic stimulation also increases the frequency of mESCs, particularly toward the end of a train and after the train of stimuli. In contrast, these mutants do not respond to an elevation of cAMP induced by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP (Yoshihara, 1999).

To investigate a possible involvement of synaptic machinery in Drosophila visual system development, the effects of a loss of function of neuronal synaptobrevin (n-syb), a protein required for synaptic vesicle release, were studied. Expression of tetanus toxin light chain (which cleaves neuronal synaptobrevin) and genetic mosaics were used to analyze neuropil pattern formation and levels of selected neural adhesion molecules in the optic lobe. Targeted tetanus toxin light chain (TeTxLC) expression in the developing optic lobe results in disturbances of the columnar organization of visual neuropils and of photoreceptor terminal morphology. IrreC-rst immunoreactivity in neuropils is increased after widespread expression of toxin. In photoreceptors, targeted toxin expression results in increased Fasciclin II and chaoptin but not IrreC-rst immunoreactivity. Axonal pathfinding and programmed cell death are not affected. In genetic mosaics, patches of photoreceptors that lack neuronal synaptobrevin exhibit the same phenotypes observed after photoreceptor-specific toxin expression. These results demonstrate the requirement of neuronal synaptobrevin for regulation of cell adhesion molecules and development of the fine structure of the optic lobe. A possible causal link to fine-tuning processes that may include synaptic plasticity in the development of the Drosophila CNS is discussed (Hiesinger, 1999).

The finding of an onset of n-syb expression in the first half of pupation poses the question of whether synapses actually start to function so early during optic lobe development. Neuronal activity plays a major role during vertebrate visual system development. A critical period of 1 d after eclosion has been demonstrated for experience-dependent developmental plasticity in the Drosophila. It has not yet been shown whether synaptic plasticity in the Drosophila CNS extends to pupation or whether neurotransmitters are released before any form of neuronal activity. Assuming the involvement of such processes, the following time scale would be expected: first, expression and localization of proteins of the vesicle release machinery; second, release of neurotransmitter independent or dependent on spontaneous activity; and third, release of neurotransmitter dependent on evoked activity. Given the early immunoreactivity of specific synaptic vesicle cycle proteins such as n-syb and synaptotagmin before P + 25% (25% through the pupal period), the synaptic vesicle cycle appears to be available for more than half of pupal development before first evoked photoreceptor responses occur at P + 82%. Morphological analysis has revealed a brief interval of intense synapse formation in the lamina of Musca starting ~P + 62% and peaking at P + 74%. Although this time window does not necessarily correspond to the first occurrence of synapses in the optic lobe of Drosophila, and the heterogeneity of optic lobe neurons should also be taken into consideration, it may indicate that n-syb is expressed long before synapses are morphologically recognizable (Hiesinger, 1999 and references).

Apparently, not all processes between target selection and the establishment of functional connectivity are yet known. The demonstration of the dependence of neuropil patterning on NO release shows a process of terminal development in a similar time window as the neuropil patterning defects observed in the current study. With regard to the current study, one possibility would be the involvement of n-syb in the release of neurotransmitters or other factors before or during synapse development. Li (1997) observed that histamine is synthesized in photoreceptors extending from cultured imaginal disks. Histamine or other substances released by growth cones after arrival in their target layers might exert functions necessary for the establishment of a regular terminal pattern (Hiesinger, 1999).

Lack of functional n-syb has no obvious influence on target selection and the development of largely overlapping terminals of R7 and R8 cells. In contrast, further development of terminal fine structure between P + 25 and P + 50% is significantly disturbed, indicating its involvement in a fine-tuning process. This early onset of n-syb function shows that either n-syb is involved in nonsynaptic processes taking place soon after target recognition, or synapses form earlier in the Drosophila optic lobe than is generally believed. Because this time window occurs significantly before the observed upregulation of Fasciclin II in active toxin-expressing photoreceptors, the morphological changes do not depend on this CAM (Hiesinger, 1999).

Cell adhesion molecules play multiple roles during optic lobe development. Most fruitfully investigated are functions during axon guidance and target recognition and synaptic plasticity. The finding of increased Fasciclin II immunoreactivity under conditions of blocked neurotransmitter release corresponds to previous studies that have shown the opposite effect with an opposite approach: apCAM is downregulated after application of serotonin, and synaptic Fasciclin II is reduced in mutants with abnormally high neuronal activity. Although it has been demonstrated that apCAM is downregulated via endocytosis, the mechanism of activity-dependent Fasciclin II downregulation at the Drosophila neuromuscular junction remains unknown. Possible downregulation mechanisms to be considered include endocytosis, extracellular cleavage, and reduced transcription or translation in combination with a continuous turnover of the protein. The upregulation of two different types of CAMs (Fasciclin II and chaoptin) in the same cell type under conditions of blocked neurotransmitter release poses the question of the specificity of the mechanism. In the absence of functional n-syb, increased numbers of docked synaptic vesicles accumulate presynaptically. Assuming that this would result in a significant sequestration of membrane material and that the synaptic vesicle cycle is continuously replenished from cell surfaces carrying adhesion molecules, deactivation of n-syb could result in decreased intake of CAMs and thus increased CAM immunoreactivity. However, current understanding of the recycling mechanism in the synaptic vesicle cycle and different localization of CAM isoforms does not support this hypothesis. Alternatively, specifically CAMs on active terminals and fibers could be downregulated to serve as markers for the competence of the synapses for sprouting (Hiesinger, 1999).

In wild-type third instar larvae, Fasciclin II is found on R7 and R8 retinal axons. During parts of pupation, Fasciclin II is detectable at low levels on photoreceptor cell bodies. It is possible that Fasciclin II is never completely downregulated from R7 and R8 terminals but is mostly below threshold for visualization with confocal microscopy. Upregulation of Fasciclin II levels in photoreceptors lacking functional n-syb after P + 75% may thus be attributable to an accumulation of the protein, when its downregulation would normally occur via an n-syb-dependent mechanism as part of a continuous protein turnover. The finding that IrreC-rst immunoreactivity remains unaltered in photoreceptors without functional n-syb but is increased in proximal neuropils after widespread TeTxLC expression can be interpreted in two different ways: either IrreC-rst protein is not present on photoreceptor terminals at the addressed time of pupation, or the n-syb-dependent CAM downregulation mechanism has a different molecular specificity in photoreceptors than in other optic lobe cells. During axonal pathfinding IrreC-rst is expressed on photoreceptors. In pupal stages IrreC-rst is localized to rhabdomeres but not to axons and cell bodies of photoreceptors during the second half of pupation. Because rhabdomeres are unique to this cell type and seem to be a preferred localization for IrreC-rst in photoreceptors, a cell-specific distribution that excludes terminals appears more likely than a specific CAM regulation mechanism for photoreceptors. Taken together, the results presented here clearly show a requirement of n-syb for optic lobe development. Either n-syb has a previously unknown activity-independent function, or synaptic transmission is involved in optic lobe development, or both (Hiesinger, 1999 and references).

Selective effects of neuronal-synaptobrevin mutations on transmitter release evoked by sustained versus transient Ca2+ increases and by cAMP

Synaptobrevin is a key constituent of the synaptic vesicle membrane. The neuronal-synaptobrevin (n-syb) gene in Drosophila is essential for nerve-evoked synaptic currents, but miniature excitatory synaptic currents (mESCs) remain even in the complete absence of this gene. To further characterize the defects in these mutants, an examination was carried out of conditions that stimulate secretion. Despite the inability of an action potential to trigger fusion, high K+ saline can increase the frequency of mESCs 4- to 17-fold in a Ca2+-dependent manner: the rate of fusion approaches 25% of that seen in wild-type synapses under the same conditions. Similarly, the mESC frequency in n-syb null mutants can be increased by a Ca2+ ionophore, A23187, and by black widow spider venom. Thus, the ability of the vesicles to fuse in response to sustained increases in cytosolic Ca2+ persists in the absence of this protein. Tetanic stimulation also increases the frequency of mESCs, particularly toward the end of a train and after the train of stimuli. In contrast, these mutants do not respond to an elevation of cAMP induced by an activator of adenylyl cyclase, forskolin, or a membrane-permeable analog of cAMP, dibutyryl cAMP, which in wild-type synapses causes a marked increase in the mESC frequency even in the absence of external Ca2+. These results are discussed in the context of models that invoke a special role for n-syb in coupling fusion to the transient, local changes in Ca2+ and an as yet unidentified target of cAMP (Yoshihara, 1999).

Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin

Two modes of vesicular release of transmitter occur at a synapse: spontaneous release in the absence of a stimulus and evoked release that is triggered by Ca2+ influx. These modes often have been presumed to represent the same exocytotic apparatus functioning at different rates in different Ca2+ concentrations. To investigate the mechanism of transmitter release, the role of synaptobrevin/VAMP, a protein involved in vesicular docking and/or fusion was studied. A series of mutations were examined, including null mutations, in neuronal-synaptobrevin (n-syb), the neuronally expressed synaptobrevin gene in Drosophila. Mutant embryos completely lacking n-syb form morphologically normal neuromuscular junctions. Electrophysiological recordings from the neuromuscular junction of these mutants reveal that the excitatory synaptic current evoked by stimulation of the motor neuron is abolished entirely. However, spontaneous release of quanta from these terminals persists, although its rate is reduced by 75%. Thus, at least a portion of the spontaneous 'minis' that are seen at the synapse can be generated by a protein complex that is distinct from that required for an evoked synaptic response (Deitcher, 1998).

Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects

Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, a gene encoding tetanus toxin light chain was expressed in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked, but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the olfactory escape response is reduced (Sweeney, 1995).

Identification and characterization of Drosophila genes for synaptic vesicle proteins

Proteins associated with synaptic vesicles are likely to control the release of neurotransmitter. Because synaptic transmission is fundamentally similar between vertebrates and invertebrates, vesicle proteins from vertebrates that are important for synaptic transmission should be present in Drosophila as well. This investigation describes Drosophila homologs of vamp, synaptotagmin, and rab3 that are expressed in a pattern consistent with a function in Drosophila neurotransmission. One previously reported candidate (syb), a Drosophila homolog of the vamp or synaptobrevin proteins, has been shown to be expressed at very low levels in neurons and is most abundant in the gut. A neuronal Drosophila vamp (n-syb, neuronal-synaptobrevin) is described and is localized to chromosome band 62A. Northern analysis and in situ hybridizations to mRNA indicate that the novel vamp, as well as the genes for synaptotagmin (syt) and rab3 (drab3), are all expressed in the Drosophila nervous system. These genes are widely (perhaps ubiquitously) expressed in the nervous system and there is no evidence of additional neuronal isoforms of synaptotagmin, vamp, or rab3. Immunoreactivity for synaptotagmin and vamp is located in synaptic regions of the nervous system. This distribution suggests that these molecules are components of synaptic vesicles in Drosophila. The conserved structure and neuronal expression pattern of these genes indicate that they may function in processes that are required for both vertebrate and invertebrate synaptic transmission. Because of their distribution in the nervous system and because n-syb, synaptotagmin, and drab3 do not appear to be in a family of functionally redundant homologs, it is predicted that mutation of these genes will produce a profound neurological phenotype and that they are therefore good candidates for a genetic dissection in Drosophila (DiAntonio, 1993b).


REFERENCES

Search PubMed for articles about Drosophila Syb-n

Antonin, W., et al. (2000). A SNARE complex mediating fusion of late endosomes defines conserved properties of SNARE structure and function. EMBO J. 19: 6453-6464. PubMed ID: 11101518

Bhattacharya, S., et al. (2002). Members of the synaptobrevin/vesicle-associated membrane protein (VAMP) family in Drosophila are functionally interchangeable in vivo for neurotransmitter release and cell viability. Proc. Natl. Acad. Sci. 99: 13867-13872. PubMed ID: 12364587

Broadie, K., et al. (1995). Syntaxin and synaptobrevin function downstream of vesicle docking in Drosophila. Neuron. 15: 663-673. PubMed ID: 7546745

Deitcher, D. L., et al. (1998). Distinct requirements for evoked and spontaneous release of neurotransmitter are revealed by mutations in the Drosophila gene neuronal-synaptobrevin. J. Neurosci. 18(6): 2028-39. PubMed ID: 9482790

DiAntonio, A., et al. (1993b). Identification and characterization of Drosophila genes for synaptic vesicle proteins. J. Neurosci. 13(11): 4924-35. PubMed ID: 8229205

Galli, T., et al. (1995). v- and t-SNAREs in neuronal exocytosis: a need for additional components to define sites of release. Neuropharmacology 34: 1351-1360. PubMed ID: 8606784

Haberman, A., et al. (2012). The synaptic vesicle SNARE neuronal Synaptobrevin promotes endolysosomal degradation and prevents neurodegeneration. J. Cell Biol. 196(2): 261-76. PubMed ID: 22270918

Hiesinger, P. R., et al. (1999). Neuropil pattern formation and regulation of cell adhesion molecules in Drosophila optic lobe development depend on Synaptobrevin. J. Neurosci. 19(17): 7548-7556. PubMed ID: 10460261

Hiesinger, P. R., et al. (2005). The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 121(4): 607-20. 15907473

Hiesinger, P. R., et al. (2006). Activity-independent prespecification of synaptic partners in the visual map of Drosophila. Curr. Biol. 16: 1835-1843. PubMed ID: 16979562

Li, C. and Meinertzhagen, I. A. (1997). The effects of 20-hydroxyecdysone on the differentiation in vitro of cells from the eye imaginal disc from Drosophila melanogaster. Invert. Neurosci. 3(1): 57-69. PubMed ID: 9706702

Saitoe, M., et al. (2001). Absence of junctional glutamate receptor clusters in Drosophila mutants lacking spontaneous transmitter release. Science 293: 514-7. 11463917

Sinha, R., Ahmed, S., Jahn, R. and Klingauf, J. (2011). Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses. Proc. Natl. Acad. Sci. 108(34): 14318-23. PubMed ID: 21844343

Südhof, T.C. and Rothman, J. E. (2009). Membrane fusion: grappling with SNARE and SM proteins. Science 323: 474-477. PubMed ID: 19164740

Sweeney, S. T., et al. (1995). Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14(2): 341-51. PubMed ID: 7857643

Williamson, W. R. et al. (2010a). A dual function of V0-ATPase a1 provides an endolysosomal degradation mechanism in Drosophila melanogaster photoreceptors. J. Cell Biol. 189: 885-899. PubMed ID: 20513768

Williamson, W. R., et al. (2010b). Guidance receptor degradation is required for neuronal connectivity in the Drosophila nervous system. PLoS Biol. 8: e1000553. PubMed ID: 21151882

Williamson, W. R. and Hiesinger, P. R. (2010c). On the role of v-ATPase V0a1-dependent degradation in Alzheimer disease. Commun. Integr. Biol. 3: 604-607. PubMed ID: 21331254

Yoshihara, M., et al. (1999). Selective effects of neuronal-synaptobrevin mutations on transmitter release evoked by sustained versus transient Ca2+ increases and by cAMP. J. Neurosci. 19(7): 2432-41. PubMed ID: 10087058

Yoshihara, M., Suzuki, K. and Kidokoro, Y. (2000). Two independent pathways mediated by cAMP and protein kinase A enhance spontaneous transmitter release at Drosophila neuromuscular junctions. J. Neurosci. 20(22): 8315-22. 11069938


Biological Overview

date revised: 25 March 2012

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