In a first attempt to understand Highwire function, both its mRNA and protein distribution were examined. For protein localization, a monoclonal antibody was generated against Hiw. Analysis by in situ hybridization shows that HIW mRNA is largely, if not exclusively, neural specific. Antisense probes label the ventral neurogenic region as early as stage 5. The staining persists in the neurogenic region at later stages and reaches its peak around stage 13, when both CNS and PNS neurons clearly express this transcript. After embryonic stage 14, the strength of CNS and PNS cell body expression starts to decrease. By stages 16 and 17, weak staining of neuronal cell bodies is observed throughout the CNS. However, by this stage, darker staining was also observed in a pattern within the CNS that coincides with the longitudinal axon tracts found where the synaptic neuropil forms; the same pattern was observed with four independent RNA in situ probes). This expression of mRNA is not associated with specific cell bodies, and thus it is concluded that it represents mRNA in axonal processes or synaptic regions (Wan, 2000).
Hiw protein is detectable at early embryonic stages in the CNS. In contrast to the mRNA expression, a peak of protein expression around stage 13 is not seen. Rather, antibody staining reveals that Hiw protein expression in the CNS is, by and large, constant from stage 13 through the end of embryogenesis. Within the CNS, most of the protein is expressed in the region of the longitudinal axon tracts around which the synaptic neuropil forms. No protein is seen in the CNS nerve roots, none in the peripheral motor and sensory nerves, and very little in the commissural axon pathways. Thus, Hiw expression correlates with where the synaptic neuropil forms in the CNS. Outside the CNS, the predominant location of Hiw protein expression is at NMJ synapses in the embyro. Protein expression is first observed during stage 16, just as these synapses are forming (Wan, 2000).
In third instar larvae, protein expression is observed at the NMJ and at the CNS neuropil. Type I synaptic boutons are clearly stained at all NMJs on all muscles. Weaker staining of type II boutons is detected. The synaptic staining is largely absent at the NMJ in hiw mutant alleles ND8, -9, and -69, and is greatly reduced in hiw mutant alleles hiwEMS, ND16, and ND51. In addition, a transgenic line of flies (pUAS-HIWC1-LD) was generated that ectopically expresses a fragment of Hiw protein containing the epitope used for generating the antibody. The Hiw mAb detects this Hiw protein fragment ectopically expressed in muscles or in tracheal branches, further confirming its specificity. When the horseradish peroxidase (HRP) product is overdeveloped, the antibody also faintly stains the surface of muscles and each of their attachment sites. However, it is believed that this staining is due to the recognition of an unrelated protein(s) since this staining is still present at about the same level in all of the hiw mutants (Wan, 2000).
To study the localization of Hiw at individual boutons, confocal microscopy was used. The staining is not uniform in boutons but rather is confined to patches. Double labeling experiments were performed with Hiw and other synaptic markers. Antibodies against Syt and Dlg label synaptic terminals in a rather uniform fashion. Double labeling with anti-Hiw and anti-Syt, as well as anti-Dlg, confirms that Hiw is not uniform within synaptic boutons (Wan, 2000).
To further identify the position and significance of these Hiw patches, antibodies that label active zones versus the areas around active zones, called periactive zones, were sought. The postsynaptic membrane just opposite the presynaptic active zones can be labeled with antibodies against an epitope tag on transgenic glutamate receptor subunits (MHC-myc-DGluRIIB). For double labeling with anti-Hiw, however, another active zone marker that was not a mouse monoclonal antibody was needed. The Drosophila homolog of the Pak cytoplasmic kinase is such a probe. The glutamate receptor subunit and Pak are precisely colocalized. By double labeling Hiw and Pak, it was found that most Hiw patches are nonoverlapping with Pak staining spots and form a pattern complementary to that of Pak. These data indicate that Hiw is localized to the periactive zones which surround the active zones (Wan, 2000).
A similar pattern of expression is seen for the CAM Fas II. Fas II staining is seen in nonoverlapping areas around Pak staining. The staining with both Fas II and Hiw fills the space outside the Pak staining areas, suggesting that both are localized around but not in active zones (Wan, 2000).
One additional antibody was used to confirm the distribution of Hiw protein outside active zones. Dap160 is a presynaptic membrane-associated protein that physically interacts with dynamin and is organized into an 'endocytic honeycomb' that surrounds active zones. Double labeling between Hiw and Dap160 reveals that the two proteins have largely overlapping distributions, further supporting the idea that Hiw protein is localized to subboutonic regions outside the active zones (Wan, 2000).
To examine the periactive zone localization of Hiw protein in greater detail, immuno-EM analysis was performed. Hiw protein is found to be associated with both the plasma membrane of boutons in all periactive zone areas and with clusters of vesicles outside active zones. However, active zones, T bars, and vesicles clustered at active zones are devoid of staining. Some Hiw staining is also observed associated with postsynaptic membranes opposite periactive zones, even though in situ hybridization analysis does not reveal muscle expression of hiw mRNA. In immuno-EM serial section reconstruction of boutons containing 20 active zones, the nonstaining active zones (which contained T bars and clusters of clear synaptic vesicles) measured 570 ± 50 nm, while the areas between them (the periactive zones) were all stained by the anti-HIW antibody and measured 1389 ± 144 nm. Given the variability of these measurements, the Hiw exclusion zone width of 570 ± 50 nm in the immuno-EM is in the same range as, but is a bit wider than, an independent assessment of active zone width in conventional EM of 469 ± 24 nm. It is not known whether this ~50 nm (~10%) difference on each side represents a real difference or whether the width of the active zone was simply underestimated by classic ultrastructural criteria (Wan, 2000).
Highwire is a large, evolutionarily conserved protein that is required to restrain synaptic growth and promote synaptic transmission at the Drosophila neuromuscular junction. Current models of highwire function suggest that it may act as a ubiquitin ligase to regulate synaptic development. However, it is not known in which cells highwire functions, whether its putative ligase domain is required for function, or whether highwire regulates the synapse during development or alternatively sets cell fate in the embryo. A series of transgenic rescue experiments were performed to test the spatial, structural, and temporal requirements for highwire function. Presynaptic activity of highwire is both necessary and sufficient to regulate both synapse morphology and physiology. The Highwire RING domain, which is postulated to function as an E3 ubiquitin ligase, is required for highwire function. In addition, highwire acts throughout larval development to regulate synaptic morphology and function. Finally, it is shown that the morphological and physiological phenotypes of highwire mutants have different dosage and temporal requirements for highwire, demonstrating that highwire may independently regulate the molecular pathways controlling synaptic growth and function (Wu, 2005).
Hiw is reported to localize to periactive zones, the regions surrounding but not including the active zone, in presynaptic terminals. It has been proposed that the periactive zone is a specialized domain that regulates synaptic growth because at least three proteins important for regulating synaptic growth localize there: Hiw, FasII, and Still life. However, the evidence that Hiw localizes to periactive zones comes from staining with the monoclonal antibody 6H4. 6H4 does not recognize Hiw. Synaptic staining with 6H4 persists in all hiw alleles, including excision mutants that delete the region of the protein against which the antibody was raised. In addition, 6H4 is unable to recognize misexpressed transgenic Hiw. To determine where Hiw is localized, sera were generated to 10 independent peptide and fusion protein antigens. Although a single serum was found that recognizes Hiw on an immunoblot, sera that work for immunocytochemistry were not generated. As such, the endogenous localization pattern of Hiw cannot be determined. GFP-tagged UAS-GFP-Hiw transgenes were generated that are fully capable of rescuing hiw mutants. The GFP-tagged Hiw does localize to synapses, although it does not extensively colocalize with synaptic vesicles, periactive zones, or active zones. The transgenic protein does appear to be enriched in synapses, because it is barely detectable in the preterminal axon. However, this overexpressed protein is visible in cell bodies and axon bundles. Similar results are found with GFP-tagged RPM-1, which is detected in synapses, axons, and cell bodies of C. elegans. It is concluded that Hiw is likely present at synapses (Wu, 2005).
A large number of hiw alleles have been generated, including many with stop codons. All of these nonsense alleles express truncated protein and indeed also express some full-length protein attributable to readthrough of the stop codon. This finding raises the possibility that no hiw alleles are true nulls. In fact, both screens that identified hiw alleles selected for viable mutants, which may have biased the screens against true null alleles. Two new excision mutants of hiw have been generated: one that deletes at least the N-terminal 2784 amino acids (hiwDeltaN), and another that appears to delete the C-terminal 2449 amino acids (hiwDeltaC). Although either allele could potentially express a truncated but partially functional protein, these are the first alleles that do not express any full-length protein. Like the previously identified alleles, these mutants are viable and have synaptic phenotypes that are essentially identical to a previously characterized genetic null allele, hiwND8. Therefore, the previously described hiw phenotype is likely the null phenotype (Wu, 2005).
RNA in situ analysis has demonstrated that hiw is abundantly expressed in the CNS, but that cannot exclude the possibility of lower-level expression in other tissues, such as muscle. In fact, in vertebrates, PAM has been detected in skeletal muscles. This study has addressed the problem of where hiw functions. Presynaptic hiw was found to be both necessary and sufficient for generating a morphologically and physiologically normal synapse. Postsynaptic expression has no detectable effect on synaptic development. These results are consistent with findings in worms and zebrafish. The conserved site of action for hiw supports the view that the mechanism of action of hiw may also be conserved (Wu, 2005).
It is likely that Hiw and its homologs function as E3 ubiquitin ligases. It contains a RING motif that can function as E3 ubiquitin-protein ligase in vitro, and this domain of the Hiw protein is the most highly conserved across species. In flies, hiw genetically interacts with the deubiquitinating protease fat facets, and, in worms, RPM-1 physically interacts with proteins that form an E3 ubiquitin ligase complex. In addition, mutations have been identified in flies and worms that are predicted to disrupt the RING domain; however, these mutant proteins may not be expressed. Therefore, it is unknown whether Hiw ligase activity is necessary for none, some, or all of the functions of hiw at the synapse. An hiw transgene encoding a mutant RING domain has been expressed. The mutations disrupt two conserved cysteine residues that are required for activity of RING domain ubiquitin ligases. This mutant protein is abundantly expressed, but cannot rescue any of the hiw mutant phenotype. Moreover, this RING mutant acts as a potent dominant negative. This suggests that the mutant protein is not grossly misfolded but instead likely interacts with Hiw-binding proteins. This supports the assertion that the RING domain, and ubiquitin ligase activity, is essential for hiw-dependent regulation of both synaptic morphology and transmission (Wu, 2005).
Because Hiw restrains synaptic growth, it was predicted that overexpression of Hiw could reduce synaptic complexity, generating small NMJs with few boutons. No such gain-of-function was observed, indicating that endogenous Hiw levels are not limiting for synaptic growth control. No gain-of-function phenotypes have been reported for rpm-1 either. These results are consistent with the finding that RPM-1 binds to a multiprotein ubiquitin ligase complex. Overexpressing a single member of that complex may not lead to increased function. In fact, the very weak dominant-negative phenotype caused by overexpression of wild-type Hiw suggests that excess Hiw may disrupt the stoichiometry of such a functional complex (Wu, 2005).
Attempts were made to distinguish between two models for the temporal requirement for hiw function. In hiw mutants, synaptic growth is excessive from the time of initial synapse formation. Hence, hiw may act exclusively during embryogenesis to set cell fate, programming the motoneuron for normal or excessive growth. Alternatively, hiw may function throughout development to regulate the synaptic growth rate of the motoneuron. This second model would allow for modification of synaptic growth during development and would be consistent with a function for hiw in activity-dependent synaptic plasticity. To investigate the temporal requirements for hiw, advantage was taken of the GeneSwitch Gal4 system for tissue-specific and temporally specific control of gene expression. It was found that expression of hiw after embryogenesis leads to a decrease in synaptic bouton number and a complete rescue of synaptic function. Hence, hiw not only acts in the embryo but can function throughout the larval growth period to control synaptic morphogenesis and transmission. It is noted that addition of hiw during larval development does not reverse the excess growth that occurred in the embryo and early larval stage. This may explain why similar studies in C. elegans found a requirement for rpm-1 early in synaptic development. hiw is required early in development, but the quantitative analysis of synaptic growth and function that is feasible at the Drosophila NMJ reveals that hiw also functions later in development (Wu, 2005).
Through manipulation of the timing of hiw expression, differential rescue of the morphological and physiological phenotypes of hiw was found. When hiw is expressed late, synaptic transmission is fully rescued, although there is still a twofold to threefold increase in bouton number. Hence, the physiological phenotype is not secondary to developmental defects at the synapse. The morphological and physiological phenotypes of hiw are genetically separable, because an hiw, fat facets double mutant shows a partial rescue of physiology with no rescue of morphology. However, the complete rescue of physiology by the late expression of hiw does more than show that morphology and physiology are independent: it raises the intriguing possibility that hiw functions as a short-term regulator of synaptic function. If hiw has an ongoing role in setting synaptic strength in addition to a developmental role controlling synaptic growth, it is in an ideal position to integrate and coordinate signals that regulate morphological and physiological synaptic plasticity (Wu, 2005).
Each abdominal hemisegment in the Drosophila embryo and larva has a stereotyped pattern of 30 muscles, each identifiable by its size, shape, body wall insertion position, and expression of molecular markers. About 45 motor neurons extend axons to innervate specific muscles in each hemisegment (Wan, 2000).
At the larval NMJ, there are two major types of synaptic terminals. Type I synapses have larger boutons and use glutamate as their neurotransmitter, while type II synapses have smaller boutons containing a variety of vesicles, including dense core vesicles filled with other transmitters, such as neuropeptides and biogenic amines. Type I boutons are surrounded postsynaptically by a conspicuous subsynaptic reticulum (SSR) consisting of multiple folds and invaginations of the muscle cell membrane. Type I synapses can be further divided into type Ib (big) and type Is (small) based on the sizes of their boutons. These synapses undergo dynamic growth during larval development. As the muscle increases its volume, there is a parallel increase in the number of synaptic boutons and the number and complexity of branches (Wan, 2000).
In a pilot effort to look for synaptic structural mutants, a collection of viable mutants on the X chromosome was screened, looking for structural defects at the NMJ of third instar larvae. These 230 mutant lines have various degrees of walking defects in adults. Antibodies against synaptotagmin (Syt) and Fas II were used as markers to label NMJ synaptic terminals of third instar larvae. One mutant line, hiw, has a dramatic synaptic structure phenotype. This initial hiwEMS mutant allele and all 12 subsequent hiwND mutant alleles, as well as 3 P element alleles, behave as viable recessive alleles. They are all viable as transheterozygotes with each other or with deficiencies that remove the gene (Wan, 2000).
In hiw mutants, the presynaptic boutons visualized by Syt staining are more numerous at all NMJs. Compared with wild-type synapses, hiw synapses have a greatly expanded branching pattern. The presynaptic terminal arbors contain many more Fas II-positive branches, the branches are longer, and the branch orders are increased. The synaptic area, the muscle surface area occupied by presynaptic structures, is also increase in hiw mutants. This synaptic overgrowth phenotype is 100% penetrant because expanded presynaptic arborizations and an increased number of boutons are observed at every NMJ in every segment of every mutant third instar animal (Wan, 2000).
Several aspects of the mutant phenotype were quantified. The number of type I boutons on muscles 6/7 and on muscle 4 were counted. hiw mutants have a 2-fold or greater increase in the number of boutons. After correcting for muscle area, hiw synapses also have a 2-fold increase in synaptic size compared with wild type. The combination of hiwEMS chromosome in trans over deficiencies show essentially identical phenotypes. Developmental curves of both hiw and wild-type larvae can be fitted with similar types of power functions, but hiw synapses have a larger synaptic size at first instar and grow at a higher rate during further larval stages. On average, there is a decrease of bouton size in hiw mutants. Given the dramatic increase in the number of boutons, this decrease in bouton size might reflect some limitation of one or more structural components as a result of oversprouting. Type Ib and type Is boutons still can be distinguished from one another in hiw mutants, although both are smaller than their wild-type counterparts. Type II boutons and branches seem more numerous in hiw mutants. However, this phenotype is less dramatic and more difficult to score, and thus was not quantitated (Wan, 2000).
To compare branching, three aspects of the branching pattern on muscle 12 were measured. Muscle 12 was chosen for this analysis because its innervation is relatively planar and accessible, and the branching pattern of that innervation is relatively simple in wild-type larvae, making it easier to quantitate branch number and complexity. First, the total length of this synapse (of only those branches containing type I boutons) was examined as a function of the length of the muscle. From the first larval instar stage onward, the synaptic length in hiw mutants is greater than in wild type. This relationship is maintained throughout larval development (Wan, 2000).
Second, the length of the two longest branches was measured. These branches are more than 2-fold longer in hiw mutants compared with wild type. Third, the branching pattern of this synapse was examined. The branches containing only type I boutons were divided into different groups based on their branch order and the number of branches were counted. There is a dramatic increase in the complexity of branches at hiw synapses; mutant synapses have many more branches and higher order branches. The average number of branches is 16.5 at hiw synapses on muscle 12, compared with 7.7 at the same synapse in third instar wild-type larvae. hiw synapses on muscle 12 often have several fifth order branches, something that is rarely observed in wild type (Wan, 2000).
Although the morphology of NMJ synapses is dramatically expanded in hiw mutants, the specificity of connections is not altered. The initial motor axon pathfinding and target selection appear normal. Antibodies that label both CNS and peripheral axon pathways (anti–Fas II and BP102 mAbs) reveal a normal pattern of axon pathways and a normal pattern of muscle innervation. Longer and larger presynaptic terminal branches were observed on the surfaces of muscles in hiw mutants toward the end of embryogenesis, but the specificity of contacts was normal. It appears that hiw mutants display the synaptic overgrowth phenotype from the onset of synaptogenesis during embryogenesis. This is consistent with the observation that hiw first instar larvae, even 30 min after hatching, already display a dramatic increase in bouton number (34 versus 17 in wild type at muscles 6/7) and increase in synaptic span (56% of muscle length in hiw versus 27% in wild type at muscle 12) (Wan, 2000).
To what extent are synaptic components localized normally in hiw mutant synapses? Postsynaptic glutamate receptors appear to be correctly localized just opposite active zones as determined by expression of the MHC-myc-DGluRIIA and MHC-myc-DGluRIIB transgenes in hiw mutant larvae. The presynaptic protein Syt and the synaptic CAM Fas II are expressed normally at hiw mutant synapses. Several other synaptic proteins (Discs-large [DLG], Pak, and Dap160) are also localized correctly in hiw mutants. Thus, many of the molecular events associated with synapse formation, including clustering of postsynaptic receptors and organization of presynaptic vesicles, appear normal in hiw mutants. This is consistent with the observation that hiw mutants are viable and are not severely impaired in motor behaviors (Wan, 2000).
To further examine the relationship of FasII and hiw, a test was performed to see whether overexpression of Fas II either pre- or post-synaptically can rescue the hiw mutant phenotype. Fas II overexpression cannot rescue the hiw mutant phenotype. This suggests that FasII is not downstream of hiw in the genetic control of synaptic growth (Wan, 2000).
Given the dramatic structural phenotype observed at hiw NMJ synapses, it was of interest to see whether the ultrastructure of these mutant synapses is affected. Serial sections of both hiw and wild-type boutons at muscle 6 (segment A3) of third instar larvae were examined in the electron microscope (EM). It was found that the hiw mutant synapses have active zones with T bars, clusters of synaptic vesicles, and other features that appear wild-type. The postsynaptic SSR also appears normal in size and shape. Some of these characteristics were quantitated from serial sections of 9 hiw boutons and they were compared with similar data from 18 wild-type boutons. Although hiw boutons are, on average, smaller than wild-type boutons, they nevertheless have the same relative bouton surface area per active zone. In addition, the average maximum width of individual active zones (as measured by membrane apposition, location of T bars, and clusters of synaptic vesicles) was 441 in hiw compared with 469 in wild type. Finally, the number of T bars per active zone is statistically similar. It is concluded from these data that the fundamental organization, size, and spacing of active zones in hiw mutants is relatively normal (Wan, 2000).
Given that hiw synapses, although greatly expanded, appear normal ultrastructurally, it was of interest to see whether synaptic transmission is altered at these mutant synapses. One might imagine that the expanded structure would lead to a stronger synapse. Surprisingly, it was found that these expanded synapses are actually weaker physiologically. Intracellular recordings were performed from muscle 6 (segment A3) of third instar larvae. This muscle is innervated by type I glutamatergic boutons from motor neurons RP3 and 6/7b. The postsynaptic responses to both spontaneous and evoked transmitter release were measured. The mean amplitude of spontaneous miniature junctional potentials (mEJPs), also called quantal size, is typically considered a measure of postsynaptic sensitivity to transmitter (in principle, it might also reflect the amount of neurotransmitter packed into each synaptic vesicle, although this is less likely). In contrast, the mean amplitude of the excitatory junctional potential (EJP) depends on both the postsynaptic receptor function and the amount of transmitter released from the presynaptic terminal (Wan, 2000).
The average mEJP amplitude is only slightly reduced in hiw mutants (0.65 ± 0.03 mV versus 0.91 ± 0.04 mV in wild type). However, the evoked release is greatly reduced; the mutant shows a 66% decrease compared with wild type (7.75 ± 0.86 mV versus 22.58 ± 1.73 mV, respectively). Thus, the quantal content (number of vesicles released) of the mutant synapses (13.18 ± 1.41, which is estimated by dividing the mean EJP amplitude by the mean mEJP amplitude and adjusting for nonlinear summation), is about 34% that of wild type (39.22 ± 4.64). Quantal content is an indication of presynaptic function. Since an obvious reduction in synaptic vesicle distribution was not observed in hiw mutants, the reduction in quantal content suggests more subtle defects in the release mechanism (Wan, 2000).
As a first step to gaining insight into the mechanisms of Hiw function, transgenic flies carrying different Hiw fragments were generated. One such transgene (pUAS-HIWC1-LD) contains a Hiw fragment between amino acids 2418 and 3461, fused to enhanced green fluorescent protein (EGFP). When this transgene was expressed panneurally using elav-Gal4 in an otherwise wild-type background, expanded synaptic structures were observed at the NMJ of third instar larvae. This phenotype is qualitatively similar to that of hiw loss-of-function mutants, although quantitatively it is not as severe. When the same transgene was expressed in muscles using the panmuscle 24B-Gal4 enhancer trap line, no phenotype at the NMJ was observed. This result further suggests that Hiw functions on the presynaptic side to control synaptic growth (Wan, 2000).
The covalent attachment of ubiquitin to cellular proteins is a powerful mechanism for controlling protein activity and localization. Ubiquitination is a reversible modification promoted by ubiquitin ligases and antagonized by deubiquitinating proteases. Ubiquitin-dependent mechanisms regulate many important processes including cell-cycle progression, apoptosis and transcriptional regulation. Ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila neuromuscular junction (NMJ). Neuronal overexpression of the deubiquitinating protease Fat facets leads to a profound disruption of synaptic growth control; there is a large increase in the number of synaptic boutons, an elaboration of the synaptic branching pattern, and a disruption of synaptic function. Antagonizing the ubiquitination pathway in neurons by expression of the yeast deubiquitinating protease UBP2 also produces synaptic overgrowth and dysfunction. Genetic interactions between fat facets and highwire, a negative regulator of synaptic growth that has structural homology to a family of ubiquitin ligases, suggest that synaptic development may be controlled by the balance between positive and negative regulators of ubiquitination (Diantonio, 2001).
Synaptic morphology is dynamic; once formed, synapses expand, retract, and remodel throughout life. This plasticity underlies the refinement of neuronal circuits during development and may be critical for plasticity in the adult brain. To identify molecular mechanisms regulating the morphological growth of synapses, a genetic screen was performed for molecules whose neuronal overexpression disrupts synaptic growth control at the Drosophila NMJ. A collection of flies capable of the targeted overexpression of endogenous Drosophila genes was screened and two lines, EP(3)381 and EP(3)3520, were identified whose overexpression in the nervous system leads to synaptic overgrowth. Both EP(3)381 and EP(3)3520 overexpress fat facets (faf), a deubiquitinating protease. Endogenous faf transcript is strongly and widely expressed in the developing central nervous system (CNS), demonstrating that neuronal expression of faf from the EP lines produces overexpression, not misexpression, of the transcript (Diantonio, 2001).
Anatomical analysis at the NMJ reveals that neuronal expression from both EP(3)381 and EP(3)3520 leads to an increase both in the number of synaptic boutons and in the synaptic span (the extent of the muscle covered by the synapse). This increase is not seen in flies that do not overexpress faf v or that overexpress a non-functional faf gene (elav-Gal4 crossed to EP(3)381faf-). Neuronal overexpression of faf also causes an increase in the number of synaptic branches as quantified by the number of branch points. Postsynaptic expression of faf does not affect synaptic morphology (Diantonio, 2001).
To assess the physiological consequence of neuronal faf overexpression, both spontaneous and evoked neurotransmitter release were analysed at muscle 6 of third instar larvae. Despite the greatly expanded size of the NMJ with faf overexpression, the amplitude of evoked excitatory junctional potentials (EJPs) is markedly reduced. Given that the amplitude of miniature EJPs (mEJPs) shows only a small, albeit significant, reduction, a large decrease was measured in quantal content (the number of vesicles released by the nerve) as measured by dividing the EJP amplitude by the mEJP amplitude. Neuronal overexpression of faf also leads to a reduction in the frequency of spontaneous mEJPs. The reduction in both quantal content and mEJP frequency indicates a presynaptic defect in neurotransmitter release. Other presynaptic mutants with even greater reductions in quantal content do not show a structural overgrowth, thus the anatomical phenotype described above is probably a direct consequence of faf overexpression and not a secondary consequence of this physiological phenotype (Diantonio, 2001).
faf antagonizes ubiquitin-dependent mechanisms by deubiquitinating target proteins. Alterations in synaptic structure and function owing to overexpression of faf suggest that ubiquitin-dependent mechanisms normally act to regulate the developing synapse. However, faf, a characterized deubiquitinating protease, might have other functions. To investigate the role of deubiquitination in the regulation of synaptic development, transgenic flies were generated capable of the targeted overexpression of the yeast deubiquitinating protease UBP2. This enzyme antagonizes ubiquitin-dependent mechanisms in yeast and has overlapping substrate specificity with FAF+9. Overexpression of yeast UBP2 in the nervous system of Drosophila leads to marked synaptic overgrowth and a severe reduction in presynaptic transmitter release. This phenotype is very similar to that seen with faf overexpression. Hence, antagonizing ubiquitin-dependent mechanisms by overexpression of deubiquitinating proteases markedly affects synaptic development (Diantonio, 2001).
To identify molecular pathways regulated by faf, a genetic interaction screen was performed to identify genes that enhance the faf overexpression phenotype. The X chromosome was screened for viable mutations that are lethal in combination with neuronal overexpession of faf. 7,000 chromosomes were screened and 15 lethal enhancers, 12 of which form one complementation group, were identifed. These 12 mutants are alleles of the highwire (hiw) gene and share the synaptic overgrowth phenotype described for loss-of-function hiw mutants. The hiw loss-of-function phenotypes are very similar to the faf gain-of-function phenotypes described here, with a large increase in the number of synaptic boutons, branches, and synaptic span, a small decrease in quantal size, and a large decrease in quantal content. The hiw transcript encodes a greater than 5,000 amino-acid protein that is localized to synapses and that contains a RING-H2 finger, a domain recently identified in a large family of E3 ubiquitin ligases. Hence, a potential synaptic E3 ligase was identified as a lethal enhancer of neuronal overexpression of faf. This genetic interaction provides further evidence that ubiquitination may have a central role in regulating synaptic growth and function (Diantonio, 2001).
To further investigate the genetic relationship between hiw and faf, double mutants were generated between loss-of-function alleles of hiw and faf. faf loss-of-function mutants have phenotypes in the developing eye and female germ line. In faf mutants, no defects were found in either synaptic morphology or function, possibly due to genetic redundancy between faf and one of the 17 other putative deubiquitinating proteases in the Drosophila genome. Although no phenotype was found for faf mutants in otherwise wild-type flies, in the sensitized background of a hiw mutant a requirement for faf is found. Two different loss-of-function alleles of faf both suppress the physiological phenotype of hiw, leading to a more than doubling of both quantal content and mEJP frequency. Hence endogenous faf activity acts to inhibit neurotransmitter release in a hiw background, much as increased faf activity inhibits neurotransmitter release in an otherwise wild-type background. Mutants of faf do not suppress the synaptic overgrowth seen in hiw, indicating that the physiological and morphological phenotypes in hiw are genetically separable. This suggests that either these phenotypes are mediated by different hiw substrates, or the anatomical phenotype is more sensitive to disruption of ubiquitin-dependent mechanisms. Finally, the inability to suppress the small quantal size defect suggests that this phenotype, seen in both hiw mutants and with overexpression of faf, may be a secondary consequence of synaptic overgrowth (Diantonio, 2001).
Decreases in postsynaptic activity induce a compensatory increase in presynaptic transmitter release, demonstrating that a homeostatic mechanism regulates synaptic strength during development. To assess the relationship between homeostatic and ubiquitin-dependent regulation, glutamate receptor function was disrupted in a hiw mutant. Postsynaptic expression of a dominant negative glutamate receptor (DgluRIIA-M/R) in a hiw mutant leads to an 18% decrease in quantal size. Since homeostatic compensation still occurs in a hiw mutant, it is suggested that homeostatic and ubiquitin-dependent regulation are mechanistically distinct (Diantonio, 2001).
The data presented here indicate that ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila NMJ and suggest that a balance between positive and negative regulators of ubiquitination controls the structure and function of the synapse. Antagonizing ubiquitination by the neuronal overexpression of the deubiquitinating proteases faf or yeast UBP2 leads to synaptic overgrowth and defects in neurotransmitter release. This phenotype is very similar to the loss-of-function phenotype of hiw, a putative synaptic E3 ubiquitin ligase. Gain-of-function mutants of faf enhance hiw and loss-of-function alleles of faf suppress hiw. It is proposed that hiw-dependent ubiquitination controls the level or activity of critical regulatory molecules at the synapse, and that these molecules can be deubiquitinated by faf and other deubiquitinating proteases. Ubiquitinated proteins have been identified at mammalian synapses, and ubiquitin-processing enzymes can regulate long-term potentiation and facilitation, therefore control of ubiquitination by molecules such as HIW and FAF could be a widely used mechanism for regulating synaptic growth and function (Diantonio, 2001).
The signaling mechanisms that allow the conversion of a growth cone into a mature and stable synapse are yet to be completely understood. Ubiquitination plays key regulatory roles in synaptic development and may be involved in this process. Previous studies identified the Drosophila ubiquitin conjugase bendless (ben) as important for central synapse formation, but the precise role it plays has not been elucidated. These studies indicate that Ben plays a pivotal role in synaptic growth and maturation. An incipient synapse is present with a high penetrance in ben mutants, suggesting that Ben is required for a developmental step after target recognition. Cell-autonomous rescue experiments were used to show that Ben has a presynaptic role in synapse growth. The TARGET system was harnessed to transiently express UAS-ben in a ben mutant background and a well defined critical period for Ben function was identified in establishing a full-grown, mature synaptic terminal. The protein must be present at a time point before but not during the actual growth process. Phenotypic evidence is provided demonstrating that Ben is not a part of the signal transduction pathway involving the well characterized ubiquitin ligase Highwire. It is concluded that Bendless functions as a novel developmental switch that permits the transition from axonal growth and incipient synapse formation to synaptic growth and maturation in the CNS (Uthaman, 2008).
The results from this study have given new insights into how ubiquitin system components establish functional synaptic connections. The temporal analysis of Bendless has been critical in illustrating its role as a developmental 'switch' in converting a growth cone into a mature synapse. As mentioned previously, the ben mutation is the result of a single amino acid change in the conserved catalytic core of the conjugase domain of the protein. This highlights the fact that the conjugase activity of the protein is necessary for the observed synaptic phenotype. Analysis of synaptic growth in Drosophila has primarily been done at the peripheral synapse of the NMJ. Components of the ubiquitin system, such as the ubiquitin ligase hiw, the deubiquitinating protease faf, and the synapse-associated E3 ligase PDZRN3, are known to play important roles in the growth and function of the fly NMJ. Significant studies have been performed with particular regard to the conserved family of hiw ubiquitin ligases. In Drosophila, hiw functions as a negative regulator of synapse development as mutants exhibit dramatic synaptic overgrowth at the larval NMJ. In Caenorhabditis elegans, loss of function of the hiw homolog rpm-1 results in multiple phenotypes at the NMJ as well as in the CNS. At the NMJ, some NMJs exhibit enlarged presynaptic terminals containing multiple active zones, whereas others contain underdeveloped or absent presynaptic terminals. In the worm mechanosensory circuit, the sensory neurons were found to retract synaptic branches, extend ectopic axons, and fail to accumulate synaptic vesicles, whereas some of the motor neurons exhibited phenotypes such as altered synaptic organization, branching, and overgrowth. Downstream signaling components have been isolated for both hiw and rpm-1 in Drosophila and C. elegans, respectively, and a number of conserved elements have been identified. Mutations in homologs of hiw in zebrafish and mice are also known to cause a variety of synaptic disruptions (Uthaman, 2008).
Ben and Hiw play distinct roles in synapse growth. This study has analyzed the novel roles played by these ubiquitin system components at the giant fiber system (GFS) central synapse. ben and hiw loss of function result in very different phenotypes, with ben specimens exhibiting synaptic undergrowth and hiw specimens exhibiting synaptic overgrowth. Ben function does not involve JNK, a well characterized downstream signaling partner identified for Hiw in Drosophila. It is also interesting to compare and contrast the role Hiw plays at a peripheral synapse with the role Ben plays at a central synapse. hiw mutants exhibit a presynaptic overgrowth phenotype at the NMJ, whereas ben mutants exhibit a reduction in presynaptic growth in the CNS. Also, Hiw does not localize to the nucleus and was found to regulate synaptic growth throughout development, whereas Ben has nuclear as well as cytosolic localization and only functions in a critical time period. Finally, Hiw activity is associated with the bone morphogenetic protein (BMP) retrograde signaling pathway that is known to be dependent on the retrograde motor. No evidence was found that Bendless function is dependent on the retrograde motor. All these data underline the fact that there are distinct targets for the ubiquitination cascades involving Ben and Hiw (Uthaman, 2008).
Functional neuronal circuits are established through a series of events: neurite outgrowth, axon guidance, target recognition, synapse formation, and synaptic growth and maturation. When the bendless mutant was originally characterized, Ben was thought to play an important role in either axon guidance or target recognition (Muralidhar, 1993; Oh, 1994). Analysis of the ben mutant clearly shows that Ben has an important role in synaptic growth. A number of specimens exhibit dye coupling between the GF and the motorneuron dendrite demonstrating that an incipient synapse is still formed and that the mutant phenotype arises from a failure of this immature connection to grow into a mature synapse. In addition, both gap junctional and chemical components are present at ben mutant terminals with synaptic vesicle marker localization as well as ultrastructural analyses (Uthaman, 2008).
The current view of synapse formation is that a nascent synapse can be rapidly assembled from material present in a growth cone in prepackaged vesicles and packets. After this primary rapid assembly of a nascent synapse, a secondary slower growth and maturation process takes place to result in a stable mature synapse. An insightful study on the Drosophila kinesin immaculate connections (imac) has shown it to be a permissive regulator of presynaptic maturation at the larval NMJ. Imac was found to be involved in the anterograde transport of synaptic vesicle precursors to the tip of the growth cone, an initial stage of synaptogenesis. In ben specimens, synaptic vesicles are still transported all the way down to the tip of the truncated terminal as evidenced by the localization of GFP-tagged synaptotagmin and synaptobrevin. The data strongly suggest that the ben mutant phenotype is resultant at a point after synaptic vesicular transport. Hence, it is concluded that the bendless terminal is an incipient synapse that fails to grow and mature and that Ben is a permissive regulator whose function is required for the initiation of a secondary process in presynaptic growth (Uthaman, 2008).
It is counterintuitive that, although Bendless is required for synaptic growth and maturation, the data show that it is not required during the growth process. This highlights the important role Ben plays as a developmental switch. Transient expression of UAS-ben before the growth of the presynaptic terminal was sufficient to rescue the ben phenotype anatomically and physiologically, but expression during the growth period had no effect. This suggests that Bendless is not involved in the actual growth process but rather has to be present in advance to alter signaling and initiate changes that allow growth to take place. Here it is essential to differentiate between axonal and synaptic growth, because it has been determined previously that axonal growth is unaffected in ben mutants (Muralidhar, 1993). Hence, Ben function is required to permit axonal growth to switch to synaptic growth (Uthaman, 2008).
The molecules in the signaling pathway of this novel mechanism remain to be further investigated. In conclusion, tight spatial and temporal control of synaptic connectivity in the nervous system is undoubtedly crucial to normal function. Determining how exactly Bendless regulates the formation of a mature synapse will give future novel insights into this phenomenon (Uthaman, 2008).
The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. This study used the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. The data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse (Bao, 2008).
One important finding from this study is that Lqf does not play a detectable role in SV endocytosis. Multiple lines of evidence obtained from electrophysiological, ultrastructural and optical imaging studies support this conclusion. This is the first in vivo study of Lqf or epsin 1 on SV recycling. The finding is also clearly surprising given that epsin 1 has been highly implicated to play a key role in the initiation of clathrin-coated vesicle formation and endocytosis. Does the observation reflect the special property of the fly NMJ? Lqf lacking either the ENTH domain or the clathrin-interacting C-terminus has been shown to rescue the mutant phenotype in the developing eye. These rescue results are intriguing, but they do not readily support a specific role for Lqf in CME. In particular, there are no clear mechanisms on how these truncated fragments could fulfill Lqf's clathrin-dependent functions. Interestingly, RNA interference and small interfering RNA-induced knockdown of epsin 1 fails to block the internalization of EGF receptors in HeLa cells. There is also evidence that epsin 1 functions only in clathrin-independent endocytosis. Furthermore, Lqf has been shown to be required for endocytosis of select receptors but not of all receptors. More importantly, Lqf itself is not required for receptor-mediated endocytosis. Rather, Lqf appears to signal select ligands (such as Delta/Serrate/Lag2) for internalization or recycling. Hence, these studies lend strong support to observations that Lqf does not play a significant role in CME of SVs (Bao, 2008).
It should be noted that recent studies reveal that the epsin 1-interacting protein Eps15 is required for SV recycling in both C. elegans and Drosophila. However, Eps15 is required to maintain the level of endocytotic proteins in nerve terminals. Strikingly, key endocytotic proteins such as Dynamin and Dap160 are reduced in synaptic boutons by ∼90% and ∼80%, respectively, in eps15 mutants. These observations make it difficult, if not impossible, to assign a direct role for Eps15 in CME (Bao, 2008).
Synapse development is a highly regulated process involving a large number of molecules. The first suggestion that Lqf could have a potential role in synapse development came from studies of its deubiquitinating enzyme Faf. This notion was further supported by a direct biochemical demonstration that Lqf is a specific substrate of Faf. The current studies provide the first experimental test of this hypothesis by showing that Lqf acts downstream of Faf in promoting synaptic overgrowth. This effect on NMJ growth appears to be Faf dependent as lqf mutations alone do not dramatically affect bouton numbers. It is interesting to note that neuronal overexpression of Lqf promotes bouton budding but does not mimic the exuberant synaptic overgrowth induced by overexpression of Faf. Hence, it is suggested that Lqf is necessary but insufficient for synaptic overgrowth, raising the possibility that Lqf is not the only substrate of Faf in motoneurons (Bao, 2008).
Another important finding emerging from this study is that two distinct UPS pathways may be employed at the Drosophila larval NMJ to regulate synapse growth. The Hiw/RPM-1/Phr1 proteins have a conserved role in inhibiting presynaptic development in Drosophila, C. elegans and mammals. In C. elegans and Drosophila, the substrates of RPM-1/Hiw have been shown to be MAP kinases and MAPKKK. The current study indicates that Lqf is unlikely a substrate of Hiw in conditioning synaptic growth. In contrast, this study shows that the Faf pathway is a positive regulator of synaptic growth at the NMJ in which Lqf is an essential substrate. Hence, it is suggested that Hiw and Faf/Lqf are two distinct UPS pathways that regulate synapse development in Drosophila (Bao, 2008).
However, the relationship between the Faf and Hiw pathways in synapse development is rather complex. Intriguingly, the MAPKKK Wnd is required for synaptic overgrowth mediated by both Hiw and Faf pathways. One possibility is that Wnd acts downstream of Lqf to fulfill the function of both the Hiw and the Faf pathways. However, this idea is inconsistent with the observation that unlike lqf mutants, the wnd null mutant itself has no morphological or electrophysiological defect. More importantly, wnd mutations do not suppress the transmitter release defect seen in the hiw mutant, whereas the lqf mutant does. Alternatively, it is suggested that Hiw and Faf act through two parallel pathways and that the suppression of Faf-induced overgrowth by the wnd mutation may be mediated by Fos/Jun kinase signaling. Based on the observation that overexpression of Ubp2A increases neuronal Wnd levels, it is possible that Faf may also use Wnd as a substrate for synaptic overgrowth. However, this has yet to be tested experimentally (Bao, 2008).
Recent genetic studies have revealed an interesting feature of synapse growth and function that closely depends on protein turnover by specific UPS pathways. In Drosophila, faf or lqf mutations are capable of partially suppressing the defect in transmitter release in hiw mutants. This partial suppression is specific and should not be viewed simply as a reduction of transmitter release in faf or lqf mutant backgrounds by hiw mutations. If there were no partial suppression by faf or lqf mutations, the amplitude of EJPs would be similar to that in hiw single mutants. Because faf null mutations reduce Lqf levels, it is reasonable to suggest that Lqf acts downstream of Faf to inhibit synaptic transmission in hiw mutants. Unlike the functional interactions with hiw, however, faf or lqf mutations do not affect synaptic overgrowth in hiw mutants. Differing from lqf and faf mutations, wnd mutations fully suppress synaptic overgrowth but do not affect synaptic physiology in the hiw mutant. Hence, different ubiquitin pathways can specifically dissociate synapse growth from function (Bao, 2008).
The physiological stimuli involved in such selective modulation of synapse growth and function remain to be identified. Given the conserved role of the ubiquitin-proteasome system in synaptic plasticity across animal species, the findings reported in this study may have general neurobiological implications. In particular, it is noted that the Faf homolog in mouse, Usp9x or Fam is differentially expressed in different regions of the brain. Such spatial distribution patterns may provide a means for Usp9x to locally regulate synaptic function. Importantly, Usp9x is localized at synapses, where calcium influx rapidly regulates its enzymatic activity and deubiquitination of epsin 1. Hence, Faf and Lqf/epsin 1 are good candidate mediators of activity-dependent synaptic plasticity (Bao, 2008).
Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. This study demonstrates that autophagy positively regulates development of the Drosophila larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH2-terminal kinase (bskDN). Autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin-proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, these findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity (Shen, 2009).
Autophagy involves multiple steps, including induction, autophagosome formation, fusion of autophagosomes with lysosomes, and recycling of autophagy components. Disrupting any of these steps impairs autophagy. Several highly conserved ATG genes encoding core components of the autophagy machinery have been identified in yeast. Mutations in genes, including atg1, -2, -6, and -18, have also been isolated and characterized in Drosophila. To assess the role of autophagy in NMJ development, the effects were examined of mutations in atg genes, whose normal functions span the entire process: atg1 is defective in autophagy induction, atg6 is defective in autophagosome formation, and atg2 and -18 are defective in retrieval of other ATG proteins from autophagosomes. Regardless of the step impaired, all of these atg mutants exhibited significant reduction in NMJ size. These results demonstrate that a basal level of autophagy is required to promote NMJ development (Shen, 2009).
Overexpression of atg1+ is sufficient to induce high levels of autophagy in larval fat bodies and salivary glands. If autophagy is a positive regulator of NMJ development, an increase in autophagy might enhance synaptic growth. Consistent with previous studies, panneuronal overexpression of UAS-atg1+ under the control of C155-Gal4 or elav-Gal4 drivers induced high levels of autophagy in the nervous system, as indicated by increased staining with LysoTracker, an acidophilic dye which has been used to assess autophagy by labeling acidic structures, including lysosomes. Under these conditions, bouton number increased more than twofold. To further verify that this NMJ overgrowth was caused by elevated autophagy rather than to some other effect of atg1+ overexpression, whether mutations in other atg genes suppress this phenotype was examined. For this purpose, a null allele of atg18 (atg18δ) was generated. Removal of one copy of atg18+ had no affect on NMJ growth in an otherwise wild-type background but significantly suppressed NMJ overgrowth caused by atg1+ overexpression. Removal of both copies of atg18+ conferred almost complete suppression. Therefore, NMJ overgrowth caused by atg1+ overexpression is primarily caused by elevated levels of autophagy (Shen, 2009).
As a further test, NMJ morphology was examined after feeding larvae with rapamycin, which induces autophagy by inhibiting TOR (target of rapamycin), the key negative regulator of autophagy. Wild-type larvae fed rapamycin exhibited striking NMJ overgrowth similar to that caused by overexpressing atg1+. Rapamycin-induced NMJ overgrowth was completely suppressed by mutations in atg18. Collectively, these results demonstrate that autophagy is a key positive regulator of NMJ growth (Shen, 2009).
Wairkar (2009) observed NMJ undergrowth in atg1 mutants but did not see overgrowth with atg1+ overexpression. This discrepancy likely results from the use of different UAS-atg1+ transgenes. For example, Wairkar was able to obtain only partial (~50%) rescue of NMJ undergrowth in atg1 mutants by overexpression of their UAS-atg1rescue construct, whereas this study obtained complete rescue of this phenotype (Shen, 2009).
Atg1 has several functions unrelated to autophagy. It was found that axonal transport is disrupted in atg1-null mutants, which is a result also recently reported by Toda (2008) and Wairkar (2009). In addition, Atg1 suppresses translation by inhibiting the S6K kinase (Lee, 2007; Scott, 2007) and controls active zone density by inhibiting extracellular signal-regulated kinase (ERK) signaling (Wairkar, 2009). However, several lines of evidence indicate that these functions of Atg1 are not responsible for the NMJ phenotypes observed when Atg1 activity was altered. (1) atg2 or -18 mutants exhibited similar NMJ undergrowth but did not have defects in axonal transport. Thus, in agreement with Toda (2008), it is concluded that Atg1's role in axonal transport is distinct from its function in autophagy and NMJ growth. (2) Blocking or activating translation by overexpressing a dominant-negative S6K transgene or constitutively activated S6K transgenes by elav-Gal4 driver had little affect on NMJ growth. Moreover, coexpression of any of the three constitutively activated S6K transgenes failed to suppress NMJ overgrowth caused by atg1+ overexpression. Thus, the role of Atg1 in S6K-dependent translation does not contribute to the NMJ phenotypes associated with manipulations of Atg1. (3) An ERK mutation does not affect NMJ growth. Although this ERK mutation suppresses the deficit in active zone density in atg1 mutants, it does not suppress atg1's NMJ undergrowth phenotype (Wairkar, 2009), indicating that it is not mediated by the ERK pathway. Collectively, these results demonstrate that altered levels of autophagy are primarily responsible for the effects of Atg1 on NMJ development (Shen, 2009).
NMJ overgrowth induced by autophagy is distinctive and offers potential clues about pathways that may be involved. Formation of multiple long synaptic branches containing many small diameter boutons without any hyperbudding or satellite boutons most closely resembles the hiw phenotype, suggesting that autophagy and Hiw may function through the same pathway. Recent evidence indicates that Hiw inhibits NMJ growth by down-regulating Wnd, which in turn activates a Jun kinase encoded by bsk (basket). NMJ overgrowth in hiw is suppressed by mutations of wnd and by a dominant-negative mutation of bsk (bskDN; Collins, 2006). If the phenotypic similarity between hiw and increased autophagy reflects convergence on a common pathway, autophagy-induced NMJ overgrowth should also be suppressed by wnd and bskDN. Indeed, this is what was observed in this study. These results strongly support the idea that autophagy and Hiw converge on a Wnd-dependent MAPK signaling pathway to regulate NMJ development (Shen, 2009).
If autophagy and Hiw act via a common pathway, where do they converge? As a positive regulator of NMJ growth, autophagy could promote degradation of a negative regulator. An intriguing possibility is that Hiw is the negative regulator affected by autophagy. If a decrease in Hiw levels is responsible for NMJ overgrowth when autophagy is elevated, restoration of Hiw should suppress overgrowth. This possibility was tested by coexpressing wild-type Hiw with Atg1; Atg1-mediated NMJ overgrowth was found to be significantly suppressed. This suppression is not simply an indirect consequence of the dilution of GAL4 caused by addition of a second UAS element because coexpression of UAS-nwk+ did not suppress such NMJ overgrowth. This result also shows that Nwk (Nervous wreck), another negative regulator of NMJ growth, is not an apparent target of autophagy, as predicted by differences in phenotypes. Thus, autophagy appears to regulate NMJ growth through its effects on particular presynaptic proteins, and Hiw represents a key downstream effector (Shen, 2009).
To further test whether autophagy promotes NMJ growth by limiting Hiw, one copy of hiw+ was eliminated to determine whether this further decrease in Hiw levels enhanced the effects of atg1+ overexpression. In an otherwise wild-type background, loss of one copy of hiw+ had no affect, but it significantly enhanced atg1+-induced NMJ overgrowth. The phenotype of hiw homozygotes overexpressing atg1+ was no more extreme than hiw homozygote alone. The absence of any additive or synergistic effects further supports the hypothesis that autophagy promotes NMJ development by down-regulating Hiw (Shen, 2009).
Because Hiw antibodies do not work for immunohistochemistry, Hiw was visualized using a fully functional GFP-tagged construct to test directly whether abundance of Hiw is affected by autophagy. In an otherwise wild-type background, Hiw-GFP was strongly expressed in neurons throughout the ventral ganglion and brain lobes driven by C155-Gal4, as detected by anti-GFP staining. However, in larvae co-overexpressing atg1+, the GFP signal was reduced by ~60% relative to anti-HRP staining. This result was confirmed by Western blot analysis. Reduction of Hiw-GFP is not caused by the dilution of GAL4 by the presence of a second UAS element because coexpression of UAS-myr-RFP did not affect abundance of Hiw-GFP. These results further indicate that autophagy promotes NMJ growth by down-regulating Hiw (Shen, 2009).
These results indicate that NMJ overgrowth caused by elevated autophagy is primarily caused by reduction in Hiw. Is the converse also true? Is NMJ undergrowth in atg mutants caused by elevated levels of Hiw? To address these questions, Hiw-GFP was expressed in neurons using C155-Gal4 in various backgrounds. Hiw-GFP levels were significantly elevated in atg1 and -6 mutants compared with the controls, consistent with the idea that Hiw is down-regulated by autophagy. If this increase in Hiw is a primary cause of NMJ undergrowth in atg loss-of-function mutants, eliminating Hiw should prevent this undergrowth; i.e., mutations in hiw should be epistatic to atg mutations. Thus, NMJ morphology was examined in hiw; atg2 and hiw; atg18 double mutants, and it was found that hiw was completely epistatic, demonstrating the role of elevated levels of Hiw in NMJ undergrowth of atg mutants (Shen, 2009).
A more direct test is to determine whether overexpression of Hiw can reduce NMJ size. However, this experiment is complicated because overexpression of Hiw by a relatively weak neuronal driver (elav-Gal4) does not affect NMJ size, whereas overexpression of Hiw by a strong neuronal driver (Elav-GeneSwitch) has a modest dominant-negative effect. To determine whether increased levels of Hiw can limit NMJ growth, it appears necessary to overexpress Hiw at an intermediate level. Therefore, NMJs were examined in larvae overexpressing UAS-hiw+ via C155-Gal4. C155-Gal4/+; UAS-hiw+/+ female larvae exhibited very mild NMJ undergrowth. Stronger undergrowth was observed in C155-Gal4/Y; UAS-hiw+/+ male larvae. This difference is consistent with higher levels of C155-Gal4 expression in males than in females, owing to dosage compensation. No differences were observed in NMJ growth between C155-Gal4/+ female and C155-Gal4/Y male larvae, indicating that the undergrowth phenotypes are dependent on the levels of Hiw overexpression and not on differences in gender or expression of GAL4 alone. Thus, moderate increases in Hiw levels result in NMJ undergrowth. Furthermore, the modest NMJ undergrowth in C155-Gal4/+; UAS-hiw+/+ larvae was enhanced when one copy of atg1+, -2+, or -6+ was removed. Together, these results indicate that elevated levels of Hiw account for most of the NMJ undergrowth in atg mutants. However, excess Hiw cannot fully explain NMJ undergrowth in atg mutants because NMJ undergrowth caused by Hiw overexpression is less severe than that of atg1 and -18 mutants. Thus, when autophagy is impaired, additional negative regulators may accumulate to depress NMJ growth. It is also likely that elevated levels of Hiw target proteins other than Wnd to limit synaptic growth because loss-of-function mutations of wnd do not affect NMJ development (Shen, 2009).
Because autophagy is generally thought of as a nonselective bulk degradation process, the idea that autophagy regulates NMJ growth primarily through its effects on Hiw levels seems difficult to understand at first. However, recent studies demonstrate that autophagy can also operate in a substrate-selective mode in regulating specific developmental events (Rowland, 2006; Zhang, 2009). For example, in Caenorhabditis elegans, when postsynaptic cells fail to receive presynaptic contact, GABAA receptors selectively traffic to autophagosomes (Rowland, 2006). However, the detailed mechanism of such selectivity is unknown. Zhang identified SEPA-1 as a bridge that mediates the specific recognition and degradation of P granules by autophagy in C. elegans. Thus, one possibility is that Hiw is specifically targeted to autophagosomes via a mechanism that remains to be elucidated. It is also possible that many presynaptic proteins besides Hiw are degraded by autophagy, but it is the reduction in Hiw that primarily affects NMJ size. Moreover, although the idea that autophagy regulates Hiw directly is favored, the possibility cannot be ruled out that autophagy promotes degradation of Hiw through an indirect mechanism involving the proteasome or other pathway (Shen, 2009).
In principle, autophagy could be acting on either side of the NMJ to regulate its development. Because atg1+ overexpression in muscle results in lethality at the first larval instar, it was not possible to assess whether this affects NMJ growth. Although a postsynaptic role of autophagy in NMJ development cannot be ruled out, several results suggest that the effects of autophagy are primarily presynaptic: neuronal expression of UAS-atg1+ is sufficient to completely rescue the NMJ undergrowth in atg1 mutants, the Hiw-Wnd pathway functions presynaptically (Wu, 2005; Collins, 2006), and hiw is completely epistatic to autophagy for NMJ growth (Shen, 2009).
Autophagy is of particular interest as a regulator of synaptic growth because it is triggered in response to many environmental cues. These results demonstrate that decreasing or increasing autophagy from basal levels results in corresponding effects on synaptic size. Thus, autophagy is perfectly positioned to link environmental conditions with synaptic growth and plasticity. As such, it is intriguing to speculate on a role for autophagy in learning and memory (Shen, 2009).
Bao, H., Reist, N. E. and Zhang, B. (2008). The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling. Traffic 9(12): 2190-205. PubMed Citation: 18796008
Burgess, R. W., Peterson, K. A., Johnson, M. J., Roix, J. J., Welsh, I. C. and O'Brien, T. P. (2004). Evidence for a conserved function in synapse formation reveals Phr1 as a candidate gene for respiratory failure in newborn mice. Mol. Cell. Biol. 24: 1096-1105. 14729956
Collins, C. A., Wairkar, Y. P., Johnson, S. L. and DiAntonio, A. (2006). Highwire restrains synaptic growth by attenuating a MAP kinase signal. Neuron 51(1): 57-69. 16815332
Diantonio, A., et al. (2001). Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412: 449-452. 11473321
D'Souza, J., Hendricks, M., Le Guyader, S., Subburaju, S., Grunewald, B., Scholich, K. and Jesuthasan, S. (2005). Formation of the retinotectal projection requires Esrom, an ortholog of PAM (protein associated with Myc). Development 132(2): 247-56. 15590740
Guo, Q., Xie, J., Dang, C. V., Liu, E. T. and Bishop, J. M. (1998). Identification of a large Myc-binding protein that contains RCC1-like repeats. Proc. Natl. Acad. Sci. 95: 9172-9177. 9689053
Hirai, S., et al. (2002). MAPK-upstream protein kinase (MUK) regulates the radial migration of immature neurons in telencephalon of mouse embryo. Development 129: 4483-4495. 12223406
Hirai, S., et al. (2005). Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo. Gene Expr. Patterns 5: 517-523. 15749080
Le Guyader, S., Maier, J. and Jesuthasan, S. (2005). Esrom, an ortholog of PAM (protein associated with c-myc), regulates pteridine synthesis in the zebrafish. Dev. Biol. 277(2): 378-86. 15617681
Li, H., Kulkarni, G. and Wadsworth, W. G. (2008), RPM-1, a Caenorhabditis elegans protein that functions in presynaptic differentiation, negatively regulates axon outgrowth by controlling SAX-3/robo and UNC-5/UNC5 activity. J. Neurosci. 28(14): 3595-603. PubMed Citation: 18385318
Liao, E. H, Hung, W., Abrams, B. and Zhen, M. (2004) An SCF-like ubiquitin ligase complex that controls presynaptic differentiation. Nature 430: 345-350. 15208641
McCabe, B. D., et al. (2004). Highwire regulates presynaptic BMP signaling essential for synaptic growth. Neuron 41(6): 891-905. 15046722
Muralidhar, M. G. and Thomas, J. B. (1993). The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 11(2): 253-66. PubMed Citation: 8394720
Murthy, V., et al. (2004). Pam and its ortholog Highwire interact with and may negatively regulate the TSC1.TSC2 complex. J. Biol. Chem. 279: 1351-1358. PubMed Citation: 14559897
Nakata, K., et al. (2005). Regulation of a DLK-1 and p38 MAP kinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120: 407-420. 15707898
Oh, C. E., McMahon, R., Benzer, S. and Tanouye, M. A. (1994). bendless, a Drosophila gene affecting neuronal connectivity, encodes a ubiquitin-conjugating enzyme homolog. J. Neurosci. 14(5 Pt 2): 3166-79. PubMed Citation: 8182464
Rawson, J. M., Lee, M., Kennedy, E. L. and Selleck, S. B. (2003). Drosophila neuromuscular synapse assembly and function require the TGF-beta type I receptor saxophone and the transcription factor Mad. J. Neurobiol. 55: 134-150. 12672013
Schaefer, A. M., Hadwiger, G. D. and Nonet, M. L. (2000). rpm-1, a conserved neuronal gene that regulates targeting and synaptogenesis in C. elegans. Neuron 26: 345-356. 10839354
Shen, W. and Ganetzky, B. (2009). Autophagy promotes synapse development in Drosophila. J Cell Biol. 187(1): 71-9. PubMed Citation: 19786572
Toda, H., et al. (2008). UNC-51/ATG1 kinase regulates axonal transport by mediating motor-cargo assembly. Genes Dev. 22: 3292-3307. PubMed Citation: 19056884
Uthaman, S. B., Godenschwege, T. A. and Murphey, R. K. (2008). A mechanism distinct from highwire for the Drosophila ubiquitin conjugase bendless in synaptic growth and maturation. J. Neurosci. 28(34): 8615-23. PubMed Citation: 18716220
Wairkar, Y. P., et al. (2009). Unc-51 controls active zone density and protein composition by downregulating ERK signaling. J. Neurosci. 29: 517-528. PubMed Citation: 19144852
Wan, H. I., et al. (2000). Highwire regulates synaptic growth in Drosophila. Neuron 26: 313-329. 10839352
Wu, C., Wairkar, Y. P., Collins, C. A. and DiAntonio, A. (2005). Highwire function at the Drosophila neuromuscular junction: spatial, structural, and temporal requirements. J. Neurosci. 25(42): 9557-66. 16237161
Wu, C., Daniels, R. W. and Diantonio, A. (2007). DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth. Neural Develop. 2:16. PubMed citation: 17697379
Zhen, M., Huang, X., Bamber, B. and Jin, Y. (2000). Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exhanger with a RING-H2 finger domain. Neuron 26: 331-343. 10839353
date revised: 15 October 2011
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