comatose and NEM-sensitive fusion protein 2
One of the most significant morphogenic events in the development of Drosophila is the elongation of imaginal discs during puparium formation. This macroscopic event is accompanied by the formation of Golgi stacks from small Golgi larval clusters of vesicles and tubules that are present prior to the onset of disc elongation. The fly steroid hormone 20-hydroxyecdysone triggers both the elongation itself and the formation of Golgi stacks. Using mRNA in situ hybridisation, it has been shown that ecdysone triggers the upregulation of a subset of genes encoding Golgi-related proteins (such as dnsf1, dsec23, Syx5, and drab1) and downregulates the expression of others (such as dergic53, dbeta'COP, and drab6). The transcription factor Broad-complex, itself an "early" ecdysone target, mediates this regulation. The ecdysone-independent upregulation of dnsf1 and dsnap prior to the ecdysone peak leads to a precocious formation of large Golgi stacks. The ecdysone-triggered biogenesis of Golgi stacks at the onset of imaginal disc elongation offers the exciting possibility of advancing understanding of the relationship between gene expression and organelle biogenesis (Dunne, 2002).
Syntaxin 5 is a Golgi-localized SNARE protein that has been shown to be required for ER-Golgi traffic in yeast (Dascher, 1994) and Golgi reassembly following cell division in mammalian cells (Rabouille, 1998). The Drosophila ortholog, Syx5, like its mammalian and yeast counterparts is localized to the Drosophila Golgi and binds to alpha-SNAP. Null mutations in Syx5 are larval lethal and demonstrate impaired transport of vesicles through the secretory pathway. A hypomorphic allele of Syx5 results in impenetrant lethality, and escaping adult flies are male sterile. The male sterility results both from failure of germ cells to complete cytokinesis and from defects in spermatid elongation and maturation. Together, these results show that Syx5 is required for the proper function of the Golgi apparatus and that an efficiently functioning Golgi apparatus is required for the steps leading to the completion of cytokinesis and formation of mature sperm (Xu, 2002).
A predicted function of a SNARE protein is its ability to interact with other members of the SNARE pathway. The phenotype that results from the overexpression of a dominant-negative form of Drosophila NSF2 at the developing wing margin has been described. To determine whether Syx5 interacts genetically with NSF2, a single copy of a null mutation in Syx5 was introduced into the flies expressing dominant-negative NSF2 along the wing margin; a significant enhancement of wing notching was seen. This enhancement was as strong as any of the other known interactors identified previously and provides further evidence that Syx5 functions as a SNARE in vivo (Xu, 2002).
In situ hybridization techniques reveal expression in the nervous system during embryogenesis and in several imaginal discs and secretory structures in the larvae. Developmental modulation of dNSF-2 expression suggests that quantitative changes in the secretory apparatus are important in histogenesis (Boulianne, 1995).
The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. A Drosophila temperature-sensitive paralytic mutation in syntaxin has been isolated that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses (Littleton, 1998).
The SNARE hypothesis has been proposed to explain both constitutive and regulated vesicular transport in eukaryotic cells, including release of neurotransmitter at synapses. According to this model, a vesicle targeting/docking complex consisting primarily of vesicle- and target-membrane proteins, known as SNAREs, serves as a receptor for the cytosolic N-ethylmaleimide-sensitive fusion protein (NSF). NSF-dependent hydrolysis of ATP disassembles the SNARE complex in a step postulated to initiate membrane fusion. While features of this model remain tenable, recent studies have challenged fundamental aspects of the SNARE hypothesis, indicating that further analysis of these components is needed to fully understand their roles in neurotransmitter release. This issue was addressed by using the temperature-sensitive Drosophila NSF mutant comatose (comt) to study the function of NSF in neurotransmitter release in vivo. Synaptic electrophysiology and ultrastructure in comt mutants have recently defined a role for NSF after docking in the priming of synaptic vesicles for fast calcium-triggered fusion. An SDS-resistant neural SNARE complex, composed of the SNARE polypeptides Syntaxin, n-Synaptobrevin, and SNAP-25, accumulates in comt mutants at restrictive temperature. Subcellular fractionation experiments indicate that these SNARE complexes are distributed predominantly in fractions containing plasma membrane and docked synaptic vesicles. Together with the electrophysiological and ultrastructural analyses of comt mutants, these results indicate that NSF functions to disassemble or otherwise rearrange a SNARE complex after vesicle docking and that this rearrangement is required to maintain the readily releasable pool of synaptic vesicles (Tolar, 1998).
N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, electrophysiological and ultrastructural analyses were performed in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses reveal that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures reveals a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, transmission electron microscopy was used to examine the distribution of vesicles within synaptic terminals, and a marked accumulation of docked vesicles at restrictive temperatures was observed in comt. Together, the results define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion (Kawasaki, 1998).
The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Work at neuromuscular synapses in the temperature-sensitive NSF mutant comatose (comt) has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway was investigated. These central synapses include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produces wild-type responses at both 20°C and 36°C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the 'long latency pathway') reveals a striking difference between wild type and comt at 36°C. Repetitive stimulation of the long latency pathway leads to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase that are observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release observed at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1 (Kawasaki, 1999).
The temperature-induced paralysis of comatose mutants of Drosophila is suggestive of a function for NSF in the CNS. Mutations in the para gene encoding the subunit of the voltage-gated sodium channel also result in a similar phenotype. Paralysis in comt flies is activity-dependent, and in the doubly mutant comt;para flies comt-like paralysis does not set in until the effects of para are reversed by shifting to permissive temperatures. During recording from the thoracic flight muscles, it was observed that comt flies show a burst of spontaneous activity at restrictive temperature. This has been reported earlier as a unique characteristic of comt paralysis. The comt;para double mutant shows this burst of activity not at restrictive but only on shifting back to permissive temperature. The unusual behavior and electrophysiology of the doubly mutant flies reported here indicates a role for NSF in synaptic vesicle cycling (Sanyal, 1999).
The N-ethylmaleimide-sensitive fusion protein (NSF) has been implicated in vesicle trafficking in perhaps all eukaryotic cells. The Drosophila comatose (comt) gene encodes an NSF homolog, dNSF1. Work with temperature-sensitive (TS) paralytic alleles of comt has revealed a function for dNSF1 at synapses, where it appears to prime synaptic vesicles for neurotransmitter release. To further examine the molecular basis of dNSF1 function and to broaden the analysis of synaptic transmission to other gene products, a genetic screen was performed for mutations that interact with comt. Four mutations that modify TS paralysis in comt are described, including two intragenic modifiers (one enhancer and one suppressor) and two extragenic modifiers (both enhancers). The intragenic mutations will contribute to structure-function analysis of dNSF1 and the extragenic mutations identify gene products with related functions in synaptic transmission. Both extragenic enhancers result in TS behavioral phenotypes when separated from comt, and both map to loci not previously identified in screens for TS mutants. One of these mutations is a TS paralytic allele of the calcium channel 1-subunit gene, cacophony (Dellinger, 2000).
Identification of cacTS2 as a modifier of comt raises a number of interesting issues. The original cacophony mutant (now known as cacS) was named on the basis of an aberrant male courtship song. The courtship song is produced by a patterned beating of the wings, and this pattern as well as the wing beat amplitude are altered in cacS mutants. The finding that cac-encoded calcium channels function in neurotransmitter release suggests that impairment of central synapses may contribute to altered song patterning in cacS. Given that cac-encoded alpha1-subunits function at flight muscle neuromuscular synapses, peripheral synaptic defects may contribute to the song phenotype as well. A second issue is whether the genetic interaction of cacTS2 and comt reflects direct or indirect interactions of the encoded gene products. Electrophysiological analysis indicates that the cac-encoded alpha1-subunit mediates fast neurotransmitter release and that dNSF1 functions in maintaining the readily releasable pool of synaptic vesicles. Thus the observed genetic interaction may reflect simply that both the comt and cac gene products function in neurotransmitter release. Alternatively, the genetic interaction may result from the well-characterized biochemical interactions of SNAREs with both NSF and calcium channels. While this issue remains unresolved, two observations favor the former possibility. (1) No sequence homology has been detected between the cac-encoded alpha1-subunit and SYNPRINT sequences thought to mediate direct interactions with other synaptic proteins. (2) Preliminary synaptic electrophysiology in cacTS2 comtST17 double mutants is consistent with independent actions of comt and cac mutations in neurotransmitter release (Dellinger, 2000).
Evidence that cac functions in neurotransmitter release arises from its genetic interactions with comatose (comt), coding for a homolog of the N-ethylmaleimide-sensitive fusion protein that functions in priming synaptic vesicles for fast, calcium-triggered fusion. comt mutants exhibit rapid temperature sensitive paralysis. These mutants typically develop and function normally at permissive temperature and can be shifted to restrictive temperature to examine the acute functional consequences of perturbing a specific gene product. To broaden the analysis of the biology of neurotransmitter release to other gene products functioning in synaptic vesicle trafficking, a genetic screen was conducted to identify mutations exhibiting functional interactions with comt. One enhancer of comt was determined to be a TS allele of cac and has been designated cacTS2. Electrophysiological analysis at neuromuscular synapses has revealed that neurotransmitter release in cacTS2 is markedly reduced at elevated temperatures, indicating that cac functions in synaptic transmission (Kawasaki, 2000). Notably, rescue of rapid, calcium-triggered neurotransmitter release can be achieved in comatose mutants by neural expression of a single cDNA containing a subset of alternative exons and lacking any conserved synaptic-protein interaction sequence (Kawasaki, 2002).
Drosophila Nsf1 is most abundant in the nervous system and can be detected in larval and adult CNS. Subcellular fractionation has revealed that dNSF-1 is enriched in a vesicle fraction along with the synaptic vesicle protein Synaptotagmin. comt flies maintained at the non-permissive temperature rapidly accumulate sodium dodecyl sulfate (SDS)-resistant SNARE complexes at the restrictive temperature, with concomitant translocation of dNSF-1 from cytosol and membrane fractions into a Triton X-100 insoluble fraction. The long recovery of comt flies after heat shock induced paralysis correlates with the irreversibility of this translocation. Interestingly, while dNSF-1 also translocates in comtTP7 larvae, there is no associated neurophysiological phenotype at the neuromuscular junction or accumulation of SDS-resistant complexes in the CNS. Together, these results suggest that dNSF-1 is required for adult neuronal function, but in the larval NMJ, this function may be maintained by other isoforms (Mohtashami, 2001).
NSF is a cytosolic ATPase implicated in a variety of cellular functions including synaptic vesicle exocytosis. A lethal mutation in the Drosophila homolog of NSF (dNSF1), is reported in this study. Lethality staging and rescue experiments with the wild type dNSF1 transgene show that NSF1 is critically required during early larval stages and during late pupariation. Lethality in larval stages is associated with defects in neurogenesis as evidenced by an overall reduction in synapse size and synapse branching. Moreover, escaper adults, though showing abnormal seizure-like paralytic behavior, are normal in terms of synaptic transmission as assayed by electroretinograms. Taken together, these data indicate a role for NSF in neural growth and branching in addition to its documented role in synaptic transmission (Sanyal, 2001).
Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, a genetic analysis was undertaken of comatose (dNSF-1) in Drosophila. Characterization of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Genetic-interaction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire;comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles (Littleton, 2001).
To characterize the role of NSF-1 in synaptic transmission, a series of new comt mutations were isloated using two approaches. The first approach was to screen for X-linked temperature-sensitive paralytic mutants. Approximately 150,000 males containing an ethyl methanesulfonate (EMS)-mutagenized X-chromosome were tested for reversible temperature-dependent paralysis at 38°C. Three mutations failed to complement comt and thus represented new alleles. Three additional temperature-sensitive alleles of comt were identified in independent screens. Together with three previously known comt mutations (ST17, TP7, and ST53), nine conditional alleles of NSF-1 were characterized. A second screen that permitted isolation of new comt alleles -- even if they were associated with a lethal phenotype -- also was carried out. This screen was based on the observation that flies heterozygous for a conditional comt allele and a deletion that removes the gene are viable and manifest temperature-sensitive paralysis. Males were mutagenized with either EMS or gamma-rays and crossed to homozygous comtST53 virgin females. Approximately 10,000 heterozygous F1 females were collected from these crosses and tested for temperature-sensitive paralysis at 38°C. Four new lethal alleles and one viable allele of comt were identified in this screen. Two additional independent recessive lethal comt alleles were included in this analysis (Littleton, 2001).
To determine the underlying amino acid changes that cause the comt mutant phenotypes, the ORF of each allele was sequenced. All but one of the temperature-sensitive paralytic mutants altered highly conserved amino acids within the D1 domain, a region required for NSF-mediated disassembly of the SNARE complex. comt alleles in this category include ST17 (G274E), JN17 (G387S), DN47 (E394K), TP7 (P398S), X238 (N418I), DN48 (A447V), X91 (A448T), and ST53 (S483L). One lethal mutation, LED, maps to the D1 domain and causes a G to S substitution at amino acid 443. The remaining comt temperature-sensitive paralytic mutation was DN34, which alters a highly conserved Gly residue in the N-domain of NSF (G58D), a region required for SNAP-mediated binding to the SNARE complex. Analysis of the crystal structure of the N terminus of NSF reveals that Gly-58 lies in the first loop of the double-psi ß-barrel (DPBB) motif. The DPBB motif is common in many proteins and is often important for substrate binding. A third class of comt alleles alters the D2 domain of NSF, a region required for assembly of NSF into functional hexamers. Three lethal comt mutants lie in this domain, including 1121 (P494L), 133 (E606K), and CLP, which has a 1-bp deletion that shifts the reading frame 36 aa upstream of the carboxyl terminus and leads to a premature stop codon. One viable allele, comtG4, also disrupts the D2 domain and results in a 3-aa deletion/2-aa substitution (VQQ to GS at amino acids 525-527) near the ATP-binding site in the D2 domain. comtG4 mutants are only weakly temperature sensitive, but show a substantial reduction in lifespan with progressive weakness and lack of coordination. Because the three-dimensional structures of the D1 and D2 domains of NSF are likely to be highly conserved, the sites of the comt D1 and D2 mutants were modelled on the crystal structure of NSF-D2. A large number of residues mutated in the temperature-sensitive mutants reside in a loop region that has been postulated to serve as a lever that imparts force to the SNARE complex during ATP hydrolysis. This region of the AAA domain also has been shown to undergo conformational shifts during the ATP-binding/hydrolysis cycle in the NSF-related protein, p97. The remaining conditional alleles cluster near the ATP-binding/hydrolysis site. Of the D2 domain alleles, comt133 alters a conserved glutamate in the ATP-hydrolysis domain, indicating that this mutation is likely to abolish hexamerization of NSF (Littleton, 2001).
To characterize further the requirements for NSF-1 in vesicle trafficking, both the temperature-sensitive and lethal comt mutants were analyzed in more detail. Most comt temperature-sensitive mutations paralyze within several minutes at 38°C, similar to alleles described. Although the comtDN34 allele displays temperature-sensitive paralysis at 38°C, it requires a longer incubation time (6-7 min) and shows residual locomoter activity during longer 38°C incubations. The delayed paralysis in comtDN34 mutants correlates with its unique molecular defect in the N-terminal domain. All temperature-sensitive comt alleles show a dramatic accumulation of SDS-resistant SNARE complexes compared with wild type, suggesting an essential role for NSF in the disassembly of SNARE complexes. Each comt temperature-sensitive mutant also blocked vesicle-trafficking in Drosophila photoreceptors at 38°C, as indicated by a loss of the on/off synaptic events in electroretinogram recordings from the retina. In contrast to the temperature-sensitive paralysis of the conditional alleles, complete removal of NSF-1 results in lethality. Two lethal alleles identified result in premature stop codons that cause truncations of NSF-1. comt191 truncates in the middle of the D1 domain and is likely to be a null allele. comtG8 truncates at the end of D1. These mutations can be rescued with a free duplication carrying a wild-type comt allele or with a heat-shock driven NSF-1 transgene, indicating the lethal phenotypes are specifically associated with loss of NSF-1 activity. Null mutants died during embryonic and larval stages, although a small percentage survived to pupae (Littleton, 2001).
The accumulation of SNARE complexes in the temperature-sensitive paralytic mutants suggests these mutations disrupt the ATPase activity or subsequent force generation required for SNARE-mediated disassembly. Consistent with this model, the altered NSF protein in comtST17 mutants has been biochemically demonstrated to have a temperature-dependent block in SNARE disassembly in vitro. However, it is unknown where NSF-mediated SNARE disassembly is required during the synaptic-vesicle cycle: before docking, after docking, during fusion, or after exocytosis. To determine the stage in the synaptic-vesicle cycle where NSF is required, the comtST17 mutation was studied in more detail. Advantage was taken of two other mutations that allow specific stages in vesicle trafficking to be blocked. shiTS1 is a temperature-sensitive mutation in dynamin that blocks synaptic-vesicle endocytosis. paraTS1 is a temperature-sensitive mutation in the alpha-subunit of the sodium channel that blocks action potential propagation and subsequent evoked-vesicle fusion. Double mutants containing shiTS1 comtST17 or comtST17 paraTS1 were generated by recombination. If NSF is required for exocytosis of the nerve terminal pool of synaptic vesicles, it would be expected that NSF should be epistatic to dynamin in nerve terminals and lead to a block in vesicle fusion before the terminals are depleted of vesicles. If the inactivation of NSF does not block a step on the exocytotic side of the synaptic-vesicle pathway, then all of the vesicles would be capable of translocating to active zones, and of docking, priming, and fusing. Then, a depletion of synaptic vesicles in shiTS1 comtST17 double mutants would be expected similar to what is observed in shiTS1 mutants. In shiTS1 mutants incubated at 38°C, synaptic-vesicle proteins such as synaptotagmin, CSP, and synaptobrevin redistribute to the plasma-membrane fraction at 38°C when assayed on sucrose velocity sedimentation gradients. In wild type or comtST17 mutations incubated at 38°C, there is no shift in the distribution of synaptic-vesicle proteins. However, in shiTS1 comtST17 double mutants and in shiTS1 mutants incubated at 38°C, synaptic-vesicle proteins redistribute to the plasma membrane, indicating the synaptic-vesicle pool is capable of completing all stages of exocytosis, including fusion in comt mutants. To extend this biochemical analysis, an ultrastructural analysis was performed on photoreceptor nerve terminals. comtST17 mutants accumulate docked and undocked synaptic vesicles when incubated for 10 min at 38°C under constant light stimulation. In contrast, shiTS1 photoreceptor terminals are depleted of synaptic vesicles. Photoreceptor terminals of shiTS1 comtST17 double mutants show a depletion of synaptic vesicles similar to shiTS1, confirming that both the docked and reserve pool of vesicles can fuse in comt mutants (Littleton, 2001).
Next, how SNARE-complex accumulation in comtST17 mutants is affected by blocking vesicle recycling in shiTS1;comtST17 double mutants was investigated. If the large accumulation of SNARE complexes represents those complexes that assembled during one round of vesicle fusion at nerve terminals, SNARE-complex accumulation in shiTS1;comtST17 double mutants should be the same as in comtST17 alone. However, if multiple rounds of exocytosis of the vesicle pool can occur in the absence of NSF activity, and if these rounds of fusion are necessary to generate the large excess of unresolved SNARE complexes, a decrease in 7S accumulation should be seen in the double mutant, because shiTS1 blocks multiple rounds of vesicular cycling. SNARE complexes were isolated from comtST17 and shiTS1;comtST17 mutants. SNARE-complex accumulation is greatly reduced in the double mutant compared with comtST17 mutants alone, suggesting that multiple rounds of vesicle cycling do occur in the absence of NSF. As expected, there is still more SNARE complex present in the double mutant than in wild type, which is consistent with the accumulation of unresolved SNARE complexes formed during a single round of exocytosis. If endocytosis could be blocked without disrupting NSF activity, it was reasoned that the accumulation of SNARE complexes should not occur in shiTS1;comtST17 mutants. This possibility was tested by taking advantage of the differential sensitivity of shiTS1 and comtST17 to temperature. Whereas shiTS1 mutants become paralyzed at 29°C, comtST17 mutants do not paralyze until 35°C. Therefore, double mutants were incubated at 29°C for 10 min to block endocytosis and to deplete the vesicle pool with NSF still active and then shifted to 38°C for 10 min to block NSF activity. Analysis of SNARE-complex accumulation with this protocol demonstrates that there is no SNARE-complex accumulation in the double mutant when the vesicle pool is depleted with NSF still active. These results suggest that NSF disassembles cis-SNARE complexes that reside in the plasma membrane as a consequence of fusion. In addition, free SNAREs that are acted upon by NSF and that are trapped in the plasma membrane in shi mutants do not spontaneously reassemble into cis-SNARE complexes once NSF activity is blocked (Littleton, 2001).
A prediction of the model for NSF function is that SNARE complexes accumulate in comt as a direct consequence of vesicle fusion and a subsequent failure in disassembly of the complex postfusion. Manipulations that block release would be predicted to block the accumulation of SNARE complexes. This hypothesis was tested by using comtST17;paraTS1 double mutants. It has been noted that paraTS1 does not block synaptic transmission in the fly retina (photoreceptors do not use para-dependent action potentials). Because the eye constitutes a significant portion of the Drosophila head and would, therefore, compromise assays in comtST17;paraTS1 double mutants, the eye, specifically the contribution of photoreceptor synapses to the SNARE-complex assays, was eliminated by expression of GMR-hid (which drives the cell-death gene hid in photoreceptor primordial cells). To avoid the cumulative effect of spontaneous fusion events contributing to the build up of SNARE complexes in this assay, SNARE accumulation was assayed at 38°C for 5 min. In flies lacking photoreceptors, paraTS1 suppresses the accumulation of SNARE complex that otherwise would occur in comt mutations incubated at 38°C. This result confirms that SNARE complexes accumulate in comt mutants as a consequence of vesicle fusion. In addition, paraTS1 also eliminates the pool of SNARE complexes normally present in wild-type flies, suggesting that assembly of SNARE complexes requires nerve activity. This finding argues against stable SDS-resistant SNARE complexes forming during synaptic-vesicle docking or priming, because paraTS1 specifically blocks action potentials. An alternative explanation for the experimental findings in comt double mutants is that loss of NSF is not rapid at the restrictive temperature but occurs slowly. Several lines of evidence argue against this interpretation. Initially, eight conditional comt paralytic mutants alter distinct amino acids in the D1 domain, yet all show delayed paralysis, suggesting that this result is a general feature of the comt phenotype. Most convincingly, however, is the behavior of comt;para double mutants. Double mutants exposed to 38°C for 5 min show a rapid recovery like para mutants alone when returned to the permissive temperature. After rapid recovery and normal behavior, these flies then suddenly reparalyze several minutes later at the permissive temperature. Because comt mutants would be completely paralyzed after 5 min at 38°C and require greater than 30 min to recover at permissive temperature, it is concluded that NSF would be inactivated in the double mutant at 38°C (Littleton, 2001).
Together, these results suggest that NSF does not function in fusion, recruitment of vesicles to active zones, docking, or priming. Rather, it is proposed that NSF specifically disassembles SNARE complexes after fusion and before endocytosis. In the absence of synaptic transmission in comtST17;paraTS1 mutants, SNARE complexes do not accumulate, and behavioral paralysis induced by loss of NSF function is delayed. Thus, in the absence of vesicle fusion, NSF is not required to prime or maintain synaptic vesicles in a fusion-competent state. Once NSF has been inactivated, vesicle release and activity continues for several minutes before paralysis occurs. In shi;comt double mutants, inactivating both dynamin and NSF results in a shi phenotype, with synapses devoid of synaptic vesicles, confirming that vesicles continue to fuse in the absence of NSF activity. Thus, a large pool of excess v-SNAREs are present and can support fusion in the absence of NSF. After these pools are exhausted, a postdocking defect in fusion occurs because of an inability to form trans-SNARE pairs. These findings complement and extend the models of NSF function provided by previous analyses of comt mutants in Drosophila. Dorsal longitudinal flight muscle recordings from temperature-sensitive comt mutants have demonstrated a reduction in synaptic current during repetitive stimulation that is accompanied by an increase in the number of docked synaptic vesicles. In the absence of repetitive stimulation, no defects in fusion are detected, confirming that vesicle fusion itself does not require NSF activity. Rather, NSF is required to restore the pool of vesicles that are readily released, likely through its ability to break apart cis-SNARE complexes and reactivate individual SNAREs for additional rounds of fusion. Thus, multiple lines of evidence support the conclusion that several rounds of vesicle fusion can continue in comt mutants before synaptic transmission is finally poisoned by a lack of fusion-competent SNAREs (Littleton, 2001).
Additional studies will be required to determine the pool size of
fusion-competent SNAREs maintained by NSF activity at different synapses. This pool size will have important implications for presynaptic plasticity, as it would determine whether modulation of
SNARE assembly/disassembly rates plays a role in controlling presynaptic output during stimulation. In fly photoreceptors and central synapses, it is estimated that this pool size is sufficient to
mediate the fusion of several rounds of the entire nerve-terminal synaptic-vesicle population. This conclusion is based on the observation that in shi;comt double mutants, there is a substantial
reduction (>2-fold) in the amount of SNARE complexes that accumulate,
compared with comt mutants alone. This observation suggests that
the large increase in assembled SNARE complexes in comt
mutants results from recycling of the vesicle pool more than once. At
adult Drosophila neuromuscular junctions, the pool of
fusion-competent SNAREs seems to be smaller, because nerve-stimulation
rates as low as 5 Hz can rapidly decrease exocytosis in comt
conditional mutants. Thus, the size of the fusion-competent pool of
SNAREs can be differentially controlled at distinct synapses. Although
it is unknown how many SNARE complexes are required for the fusion of a
synaptic vesicle, the results argue that there are sufficient v-SNAREs
present on each vesicle to suffice for several rounds of exocytosis
without being regenerated through NSF action. Likewise, there seems to
be a large pool of synaptic t-SNAREs that can function in many cycles
of fusion without requiring regeneration through NSF action.
Overexpression of the NSF adapter alpha-SNAP at crayfish neuromuscular
junctions or at the squid giant synapse results in an
increase in the pool of synaptic vesicles readily released. These
findings suggest that modification of the functional pool size of
fusion-competent SNAREs over time can alter synaptic function and may
be an important mechanism for modulating synaptic plasticity. The use
of fast FM dyes recently has defined an additional synaptic-vesicle
pathway of vesicle fusion and immediate reuse. In such cases, the ability to rapidly dissociate SNARE complexes may be essential, and NSF
activity may be rate-limiting in this rapid reuse pathway. The recent
finding that patients with schizophrenia show a substantial reduction
in NSF expression in the prefrontal cortex suggests that
disruption of SNARE-mediated disassembly rates also may be a
contributing factor in psychiatric dysfunction. Further studies into
the mechanisms that maintain fusion-competent SNAREs and accelerate NSF
activity should provide insights into presynaptic plasticity and the
machinery that modulates synaptic-vesicle fusion (Littleton, 2001).
The Drosophila larval neuromuscular system serves as a valuable model for
studying the genes required for synaptic development and function.
N-Ethylmaleimide sensitive factor (NSF) is a molecule known to be important in
vesicular trafficking; neural expression of a dominant negative form of NSF2
induces an unexpected overgrowth of the Drosophila larval neuromuscular
synapse. A genetic approach was taken to study this phenotype
by conducting a gain-of-function modifier screen to isolate genes that interact
with the overgrowth phenotype. The approach was to directly visualize the
neuromuscular junction (NMJ) using a GFP transgene and screen for suppressors
of NMJ overgrowth using the Gene Search (GS) collection of P-element insertions. Of
the 3000 lines screened, 99 lines were identified that can partially restore the
normal phenotype. Analysis of the GS element insertion sites by inverse PCR and
comparison of the flanking DNA sequence to the Drosophila genome sequence
revealed nearby genes for all but 10 of the 99 lines. The recovered genes, both
known and predicted, include transcription factors, cytoskeletal elements,
components of the ubiquitin pathway, and several signaling molecules. This
collection of genes that suppress the NSF2 neuromuscular junction overgrowth
phenotype is a valuable resource in efforts to further understand the role
of NSF at the synapse (Laviolette, 2005).
Eighteen GS lines were discovered whose candidate genes can be broadly categorized as transcriptional regulators. Several of these candidate genes are expressed in the nervous system or in some cases loss-of-function alleles are known to produce neural phenotypes, e.g., longitudinals lacking, buttonless, couch potato, hoi polloi, E2f, brain tumor, nejire, and retinoblastoma-family protein (Laviolette, 2005).
Interestingly, cyclic AMP response element-binding protein (CREB) (nejire) function has been analyzed at larval neuromuscular synapses and while the presynaptic overexpression of CREB was found to impair neurotransmitter release, it has no effect on presynaptic morphology. This study found that a GS line in position to overexpress CREB in the NSF2E/Q mutant background rescues the overgrowth phenotype, suggesting that this gene may have a role in presynaptic development (Laviolette, 2005 and references therein).
Three of the 18 transcriptional regulators, buttonless, couch potato, and hoi polloi, have been recovered in loss-of-function screens designed to uncover genes important for peripheral nervous system development. The results indicate the potential for these genes to also be involved in motor
neuron development (Laviolette, 2005).
One gene that was recovered, ovo, is not normally associated with neural
function. ovo is normally expressed in the male and female germline cells,
where it controls F-actin extension. When expressed in the eye under UAS/Gal4
control, eye bristle formation is impaired and induces ectopic extensions from
the ommatadia, suggesting that it has a strong influence on cytoskeletal
remodeling (Laviolette, 2005).
It is likely that overgrowth of the neuromuscular junction ultimately
affects the underlying cytoskeleton but it was somewhat surprising to identify
a large number of genes composing structural components of the cytoskeleton or
enzymes with the potential to regulate cytoskeletal dynamics. The structural
genes are Actin5c, moesin, fimbrin, syntrophin-like
2, Myosin binding subunit, Ptpmeg, and CG5740. Two
genes that encode proteins with enzymatic activity that have the potential to
regulate the cytoskeleton, RhoBTB and RhoGap18B, were also found (Laviolette, 2005).
Actin5C is one of two cytoplasmic actins in Drosophila and it is highly expressed in many tissues throughout development. It has been studied extensively in many processes, including spermatogenesis and dorsal closure. In the nervous system, Act5c has been recovered in a screen for changes in bristle number -- presumably a reporter of peripheral nervous system development -- and its role has been studied in glial cell function (Laviolette, 2005).
Moesin is a member of the ezrin-radixin-moesin (ERM) family of actin-binding
proteins that link actin to the plasma membrane. While a neural role for Drosophila
moesin has not previously been reported, it does have a role in growth cone motility in other neural systems, and a recent report demonstrates the role of Drosophila moesin in photoreceptor rhabdomere development (Laviolette, 2005).
Four genes involved in the ubiquitin protein degradation pathway were identified: bendless, Bruce, thread, and
Ufd1-like. The gene products of bendless, Bruce, and
thread (also known as DIAP1) are thought to act as E2 or E3
ubiquitin-conjugating enzymes. The Ufd1-like gene encodes
a transcript that is involved in ubiquitin-dependent protein degradation. Thus, the
four candidates in this category would all have the potential to enhance
ubiquitin-dependent protein degradation if their expression were to be increased (Laviolette, 2005).
Identification of these genes is an important finding in light of previous
developmental NMJ studies, which revealed that loss-of-function mutants of
highwire, whose gene product contains a RING finger domain implicated in
E3 ubiquitin ligase activity, leads to NMJ overgrowth that is very similar to
NSF2E/Q-induced overgrowth. Furthermore, transgenic
overexpression of fat facets also causes excessive NMJ overgrowth. faf encodes a deubiquitinating protease and is thought to protect
ubiquitinated proteins from degradation (Laviolette, 2005).
Therefore, hiw loss of function or faf overexpression should lead
to reduced ubiquitin-dependent protein degradation. Since the
NSF2E/Q phenotype is rescued by expression of genes that
should increase the ubiquitin degradation pathway, it is inferred that reduced
ubiquitin pathway function may also be one of the molecular dysfunctions
underlying the NSF2E/Q phenotype (Laviolette, 2005).
Three genes involved in G-protein-receptor-coupled signaling were identified:
methuseleh (mth), Frizzled 4, and AlstR. methuseleh
is a gene first identified for its effects on life span. Prior
analysis of mth at the NMJ, using hypomorphic loss-of-function mutants,
revealed no effect on the number of synaptic boutons or length of NMJ branches. The current results suggest that mth may have a previously undetected developmental role at the synapse (Laviolette, 2005).
Frizzled 4 is a Wnt receptor that is expressed in the CNS, but so far
there has been no genetic analysis of this receptor. However, a recent study
has demonstrated the involvement of Wnt signaling at the Drosophila NMJ,
showing that wingless (wg) loss of function results in fewer
boutons while presynaptic wg overexpression produces more boutons; these
effects are thought to be mediated through the Wnt receptor Frizzled 2. While it is
currently unknown if the CNS expression of fz4 is presynaptic or not,
the current result imply that there may be an autocrine component to Wnt signaling at the synapse, as there is during wing development (Laviolette, 2005).
There is no previous neural expression or mutant data for Galpha73B, a
component of a trimeric G-protein complex, and it is not presently clear how
this gene aids the restoration of NSF2E/Q neuromuscular
overgrowth (Laviolette, 2005).
Four genes with known or predicted kinase activity were also recovered:
Pka-C1, grapes, Pak3, and CG6386. Prominent in this
group is Pka-C1, which encodes cAMP-dependent protein kinase 1. Previous
studies of rutabaga, the adenylyl cyclase that produces cAMP, and
dunce, the phosphodiesterase that hydrolyzes cAMP, have shown dramatic
effects on NMJ morphology and physiology. Loss of function of either
rut or dnc leads to NMJ overgrowth, although the extent of that
effect is not as large as is seen with the NSF2E/Q allele.
Interestingly, GS9123 is inserted 252 bp upstream of the rutabaga
5'-UTR; however, the orientation of the UAS sequence appears to be in the wrong
direction to drive expression of rut (Laviolette, 2005).
PAK3 is a member of the p21-activated kinase family that has been shown in
other systems to strongly influence the cytoskeleton by way of its interactions with the small GTPases Rho and cdc42 that directly regulate actin biochemistry. Thus, the finding of PAK3 also potentially fits with observations of the
cytoskeletal components interacting with NSF2 (Laviolette, 2005).
Three genes with activity in the Ras signaling pathway were found: C3G,
PTP-ER, and CG32560. C3G is a RAS family guanine nucleotide exchange
factor that activates Ras by catalyzing exchange of GDP for GTP. PTP-ER encodes a tyrosine phosphatase that dephosphorylates Drosophila MAPK,
thereby downregulating its kinase activity. CG32560 is
predicted to be a RAS GTPase activator, which would activate GTP hydrolysis and
downregulate RAS signaling. Thus, the result of C3G appears to be in
contradiction to the results of PTP-ER and CG32560 (Laviolette, 2005).
The Spitz protein is the EGF receptor (EGFR) ligand that has been implicated in
many developmental processes, including photoreceptor axon guidance. Rolled is
the Drosophila homolog of MAP kinase and a downstream target of EGFR. MAPK has been linked to Netrin-dependent growth cone attraction through the Netrin receptor DCC (Laviolette, 2005).
Finally, another important signaling gene identified was
NetrinB. This is one of two Netrin isoforms in Drosophila that are
well known for their role in axon guidance, both in the periphery and in
central commissural axons that cross the midline. NetrinB is a secreted
molecule that can interact with two receptors: frazzled, the
DCC receptor that exerts attractive cues, and unc-5, which can
mediate repulsive cues in axon guidance. Although Drosophila Netrins
are widely known to be expressed in midline glial cells and in muscles, NetrinB
RNA and protein are also found in a number of ventral-lateral neurons in the
Drosophila embryo. Therefore,
the rescue of the NSF2E/Q overgrowth phenotype by presynaptic
expression of NetrinB may reveal an autocrine component to this signaling
pathway whereby Netrin is released from the growth cone or mature nerve
terminal and acts upon Netrin receptors there (Laviolette, 2005).
Two other interesting lines were recovered that do not fit into the above
classifications. (1) GS3062 is positioned 0.4 kb upstream of polo,
which acts as a serine/threonine kinase and hypomorphic alleles of this gene
affect larval brain development through cytokinesis defects. However, this GS
element is also 3.5 kb upstream of the soluble NSF attachment protein
(snap) gene. The SNAP protein is the cofactor that links NSF to the
SNARE complex and to AMPA-type glutamate receptors. Therefore, while snap is a very attractive candidate, it remains to be determined whether GS3062 drives
expression of polo, snap, or both (Laviolette, 2005).
(2) GS8030 was identified as a line with the potential to activate Rab5 expression. This gene is a member of the Rab small GTPase family, and Rab5 in particular is thought to be an important component in the endocytic pathway. Recently, Rab5 has also been shown to be critical for receptor tyrosine-kinase-induced actin remodeling in mouse fibroblasts and thus there is a potentially interesting link between this gene and the cytoskeleton (Laviolette, 2005).
N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity Drosophila has two NSF genes, comatose (which encodes the NSF1 protein) and NSF2, whose proteins are 80% identical and are functionally redundant. NSF1 is the predominant functional isoform in the adult fly nervous system, although NSF2 is functionally important in other tissues and throughout development. Flies that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. This study tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging a substantial reduction was found in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. It is concluded that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. These data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin (Nunes, 2006; full text of article).
Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (an acronym derived from 'SNAP (Soluble NSF Attachment Protein) REceptor', consisting of proteins that mediated vesicular fusion with the target membrane) in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila, which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. The findings establish an in vivo role for the target-Snare (t-Snare) protein SNAP-25, one part of the four helix bundle of proteins referred to as SNAPs, in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for wild-type PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, Syntaxin and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported in this study establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression (Kawasaki, 2009).
The present study further defines the in vivo roles of NSF and SNAP-25 in synaptic vesicle trafficking and their contributions to conserved properties of short-term synaptic depression. Insight is also gained into the spatial organization of activity-dependent SNARE protein cycling with respect to active zones. The findings support a model incorporating in vivo molecular mechanisms of synaptic vesicle priming as important determinants of short-term depression and maintenance of neurotransmitter release during synaptic activity. These priming mechanisms include a direct role for SNAP-25 and an indirect contribution involving NSF-dependent replenishment of active zone free t-SNAREs for subsequent vesicle priming and fusion (see Working model of SNARE protein cycling during synaptic activity). In this model, recovery after a high frequence stimulation, in a measurement called paired pulse depression (PPD), depends on a limited pool of free active zone t-SNAREs (A), which is sufficient to support formation of trans-SNARE complexes (B) and refilling of the release-ready vesicle pool. This priming process is impaired in the SNAP-25TS mutant with respect to WT. The location of the SNAP-25TS mutation within the N-terminal region of the SNARE four helix bundle is consistent with a zippering model of SNARE complex function. On train stimulation, cis-SNARE complexes accumulate in the periactive zone (PAZ), resulting in depletion of the free t-SNARE pool at the active zone (AZ). Disassembly of cis-SNARE complexes by NSF and SNAP restores active zone t-SNARES for subsequent vesicle priming and fusion events. This aspect of synaptic vesicle priming is impaired in comatose, which encodes an enzyme that facilitates priming (Kawasaki, 2009).
The findings of this study reveal a role for SNAP-25 in determining the properties of recovery from short-term depression. An underlying function for SNAP-25 in synaptic vesicle priming was inferred from the following aspects of the SNAP-25TS synaptic phenotype: (1) activity dependence as indicated by a WT initial EPSC amplitude and release-ready synaptic vesicle pool size, (2) preservation of the WT EPSC waveform, (3) loss of fast recovery in PPD, which is associated with fast refilling of the release-ready pool and exhibits a time course comparable with that of synaptic vesicle priming, (4) dependence on previous synaptic vesicle fusion, and (5) slowed refilling of the release-ready vesicle pool. Thus, although SNAP-25 is required for evoked synaptic vesicle fusion (Washbourne, 2002), the SNAP-25TS mutation can selectively affect synaptic vesicle priming. This finding is consistent with systematic structure-function analysis of SNAP-25 in adrenal chromaffin cells, which provided strong support for the zippering model of SNARE function (Sorensen, 2004). In this model, vesicle priming involves initial assembly at the N-terminal end of the SNARE four helix bundle to form a loose trans-SNARE complex. Final assembly of its C-terminal end, which may be triggered by calcium influx, is critical for vesicle fusion. Notably, the SNAP-25TS mutation is positioned within the N-terminal region of the four helix bundle, consistent with a TS impairment of trans-SNARE complex formation associated with synaptic vesicle priming. Previous studies of SNAP-25TS indicate that synaptic vesicle docking at active zones is not disrupted at larval neuromuscular synapses, and that in vivo levels of SDS-resistant (likely cis) SNARE complexes are not altered in this mutant. Finally, it will be of great interest to further investigate the relationship of SNAP-25-dependent mechanisms underlying fast and slow components of recovery. These components are thought to involve calcium-dependent regulation of kinetically distinct synaptic vesicle pools exhibiting different release probabilities (Kawasaki, 2009).
The close resemblance of comatose (NSF) and WT synapses with respect to the initial EPSC amplitude and PPD suggests that mutant comatose (NSF) initially exhibits a WT release-ready vesicle pool and release probability. The strictly activity-dependent reduction in neurotransmitter release observed in comatose (NSF) mutants requires a brief period of synaptic activity as indicated by delayed onset of the synaptic phenotype during train stimulation. The underlying mechanism appears to involve activity-dependent redistribution of plasma membrane t-SNAREs away from the active zone. Together with systematic biochemical studies of comatose (NSF) demonstrating TS accumulation of (SDS-resistant) ternary SNARE complexes on the plasma membrane, these findings suggest that SNAREs accumulate in plasma membrane cis-SNARE complexes that are not retained within the active zone (see the working model). NSF disassembly of post-fusion plasma membrane cis-SNARE complexes has been directly demonstrated in studies of yeast exocytosis and the underlying heterotypic fusion of secretory vesicles with the plasma membrane. Previous and present efforts to address this issue at Drosophila synapses, including analysis of v-SNARE distribution in the presynaptic plasma membrane. Finally, an alternative view of NSF function favoring prefusion disassembly of SNARE complexes was reported in a study employing caged NSF peptides to acutely disrupt NSF function at the squid giant synapse. However, the activity-dependent effects of the peptide on EPSC amplitude are largely consistent with the working model presented in this study, in which the timing of the NSF requirement is not constrained by the total vesicle cycling time. Although the NSF peptide was also found to slow EPSC rise and decay times, no analogous effects were observed in the present study (Kawasaki, 2009).
The adult DLM neuromuscular synapse has had an important role in analysis of TS mutations in comatose (NSF), the presynaptic calcium channel α1 subunit gene, cacophony, and the Dynamin gene, shibire. This work has revealed that several functional characteristics of adult neuromuscular synapses are distinct from those described in the larva. In the present study, comparison of glutamatergic DLM neuromuscular synapses and cerebellar CF-PC synapses revealed a surprising degree of conservation in the detailed properties of synaptic function. These synapses exhibit morphological similarities as well, including extensive branching of axons and spatial isolation of active zones. Initial characterization of short-term depression at CF-PC synapses has been followed by a series of studies progressively defining the underlying factors in greater detail. The present study provides a basis for analysis of DLM neuromuscular synapses to characterize analogous factors and their molecular determinants. Finally, the results reported in this study complement previous and ongoing studies at DLM neuromuscular synapses of the Dynamin TS mutant, shibire, which exhibits a rapid enhancement of short-term depression as observed in SNAP-25TS. Such similarities may reflect interactions of the in vivo molecular mechanisms governing synaptic vesicle endocytosis and exocytosis (Kawasaki, 2009).
Techniques to induce activity-dependent neuronal plasticity in vivo allow the underlying signaling pathways to be studied in their biological context. This study demonstrates activity-induced plasticity at neuromuscular synapses of Drosophila double mutant for comatose (an NSF mutant) and Kum (Calcium ATPase at 60A: a SERCA mutant), and presents an analysis of the underlying signaling pathways. comt; Kum (CK) double mutants exhibit increased locomotor activity under normal culture conditions, concomitant with a larger neuromuscular junction synapse and stably elevated evoked transmitter release. The observed enhancements of synaptic size and transmitter release in CK mutants are completely abrogated by: a) reduced activity of motor neurons; b) attenuation of the Ras/ERK signaling cascade; or c) inhibition of the transcription factors Fos and CREB. All of which restrict synaptic properties to near wild type levels. Together, these results document neural activity-dependent plasticity of motor synapses in CK animals that requires Ras/ERK signaling and normal transcriptional activity of Fos and CREB. Further, novel in vivo reporters of neuronal Ras activation and Fos transcription also confirm increased signaling through a Ras/AP-1 pathway in motor neurons of CK animals, consistent with results from the genetic experiments. Thus, this study: a) provides a robust system in which to study activity-induced synaptic plasticity in vivo; b) establishes a causal link between neural activity, Ras signaling, transcriptional regulation and pre-synaptic plasticity in glutamatergic motor neurons of Drosophila larvae; and c) presents novel, genetically encoded reporters for Ras and AP-1 dependent signaling pathways in Drosophila (Freeman, 2010).
This study describes a new model for activity-dependent pre-synaptic plasticity in Drosophila. In the double mutant combination of comt and Kum, sustained elevation of neural activity (potentially including seizure-like motor neuron firing under normal rearing conditions) results in the expansion of motor synapses with a concomitant increase in transmitter release. These synaptic changes are mediated by the Ras/ERK signaling cascade and the activity of at least two key transcription factors, CREB and Fos. In vivo reporter assays also directly demonstrate Ras activation and enhanced transcription of Fos in the nervous system. CK is the only genetic model of synaptic plasticity in Drosophila in which pre-synaptic plasticity has been correlated with the Ras/ERK signaling cascade. This result is especially relevant given the wide conservation of the Ras/ERK signaling cascade in plasticity and recent demonstrations of the involvement of this signaling cascade in learning behavior in flies (Godenschwege, 2004; Moressis, 2009). Significant insights into Ras mediated regulation of both synapse growth and transmitter release are also presented (Freeman, 2010).
Non-invasive methods to manipulate neural activity in select neurons continue to be an important experimental target in plasticity research. In Drosophila, combinations of the eag and Shaker potassium channel mutants have long been used to chronically alter neural activity and study downstream cellular events. In recent years, transgenic expression of modified Shaker channels has also been generated and used to alter excitability in both neurons and muscles. However, the CK model of activity-dependent plasticity was developed since in synaptic changes in CK were consistently more robust than eag Sh and core plasticity-related signaling components were activated in a predictable manner in CK mutants. Another advantage with CK is the option of acutely inducing seizures as has been used to identify activity-regulated genes. CK thus combines advantages of both eag Sh and seizure mutants, and as is shown in this study, leads to an activity-dependent increase in synaptic size and transmitter release. It is believed that this model will prove highly beneficial to the large community of researchers who investigate synaptic plasticity in Drosophila. The utility of more recent techniques (such as the ChannelRhodopsin or the newly reported temperature sensitive TrpA1 channel transgenes) to induce neural activity-dependent synaptic plasticity at Drosophila motor synapses has not been tested yet and it will be interesting to see if these afford greater experimental flexibility in the future (Freeman, 2010).
Signal transduction through the Ras cascade has been shown to affect both dendritic and pre-synaptic plasticity in invertebrate and vertebrate model systems. In mammalian neurons, Ras signaling has been linked to hippocampal slice LTP, changes in dendritic spine architecture and plasticity of cultured neurons. In this context, Ras signaling has been shown to impinge on downstream MAP kinase signaling, thus implicating a canonical signaling module already established as a mediator of long-term plasticity in vertebrates. In Drosophila, expression of a mutant constitutively active Ras that is predicted to selectively target ERK leads to synapse expansion and increased localized phosphorylation of ERK at pre-synaptic terminals. In light of these observations, tests were performed to see if Ras signaling os necessary and sufficient for synaptic plasticity in CK. The results suggest that synaptic changes in CK are driven by stimulated Ras/ERK signaling in Drosophila motor neurons, and these can be replicated by directly enhancing Ras signaling in these cells. Furthermore Ras activation was found to be sufficient to cause stable elevation in pre-synaptic transmitter release. Finally, evidence is provided to show that synaptic effects of Ras activation require the function of both Fos and CREB in motor neurons. The consistency of signaling events in CK with those observed in mammalian preparations makes this a more useful and generally applicable genetic model of synaptic plasticity (Freeman, 2010).
In vivo reporters of neural activity have been difficult to design but offer better experimental resolution and flexibility over standard immuno-histochemical or RNA in situ methods to detect changes in gene expression in the brain. Thus, a good reporter permits increased temporal and spatial resolution, the option of live imaging (for fluorescent reporters) and in the case of transcriptional reporters, better understanding of cis-regulatory elements that control activity-dependent gene expression. This paper describes two genetically encoded reporters with utility clearly beyond the current study; a Raf based reporter to detect Ras activation in neurons and an enhancer based reporter to detect transcription of Fos (Freeman, 2010).
The Ras binding domain of Raf has been used previously to detect Ras expression in yeast, mammalian cell lines, and recently in hippocampal neuron dendrites. This study used a similar strategy to model the reporter using the conserved Ras binding domain and the cysteine-rich domain (RBD + CRD) from Drosophila Raf, under the reasonable assumption that this would provide sensitive reporter activity in neurons. This is the first time that a Ras reporter has been utilized in an intact metazoan organism to measure changes in endogenous Ras activity. In addition to confirming Ras activation in CK brains, it is expected that this reporter will find widespread use in tracing Ras activation in multiple tissues through development and in response to signaling changes in the entire organism. Since the reporter is based on the GAL4-UAS system, it can be expressed in tissues of choice, limiting reporter activity to regions of interest. Indeed, the experiments with the eye-antennal imaginal disc illustrate the utility of this reporter in identifying regions of activated Ras signaling during eye development (Freeman, 2010).
The Fos transcriptional reporter is one of the very few activity-regulated reporters in existence in Drosophila and it should find broad acceptance as a tool to map neural circuits in the fly brain that show activity-dependent plasticity. The reporter believed to be reasonably accurate since it is expressed in expected tissue domains (embryonic leading edge cells, for instance), and also co-localizes extensively with anti-Fos staining in the larval brain. There are several recognizable transcription factor binding motifs that can be detected in this 5 kb region of DNA (including binding sites for CREB, Fos, Mef2 and c/EBP). Which of these transcription factors regulate activity-dependent Fos expression from this enhancer is currently unknown. However, future experiments that dissect functional elements in this large enhancer region are expected to refine and identify these regulatory elements. Such studies are likely to lead the way in the development of a new generation of neural activity reporters in the brain (Freeman, 2010).
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date revised: 5 August 2011
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